United States Patent [19]

US005696887A
United States Patent [19]
Bernstein et al.
[11] Patent Number:
[45] Date of Patent:
603577
[54] AUTOMATED TISSUE ASSAY USING
STANDARDIZED CHEMICALS AND
PACKAGES
2143205
Santa Barbara, all of Calif.
[73] Assignee: Biotek Solutions, Incorporated, Santa
Barbara, Calif.
[21] Appl. No.: 252,282
May 31, 1994
[22] Filed:
Related U.S. Application Data
[63] Continuation-in-part of Ser. No. 740,285, Aug. 5, 1991, Pat.
No. 5,355,439, and Ser. No. 218,143, Mar. 24, 1994.
[51] Int. Cl* … G06F 9/00; G06F 15/46
[52] U.S. Cl. ….…..... 395/82
[58] Field of Search ................................... 395/81, 82, 99
References Cited
[56]
U.S. PATENT DOCUMENTS
3,665,148
5/1972 Yasenchak et al. ..................... 219/125
4,087,248
5/1978 Miles .....................
4,484,293 11/1984 Minucciani et al.
.... 23/230 B
---... 364/513
4,659,971 4/1987 Suzuki et al. ......
. 318/568
4,719,087 1/1988 Hanaway .......
... 422/102
4,727,494 2/1988 Buote .........
... 364/513
4,807,152 2/1989 Lane et al. .........
. 364/513
4,835,711 5/1989 Hutchins et al. ......
. 364/513
4,843,566 6/1989 Gordon et al. .........
. 364/513
4,979,093 12/1990 Laine et al. ........
..... 364/167
4,979,128 12/1990 Seki et al. ......
... 364/513
... sºoi
5,016,170 5/1991 Pollalis et al. .
... 422/64
5,229,074 7/1993 Heath et al.
... 422?65
5,281,394 1/1994 Holub .............
5,301.261 4/1994 Poole et al. ............................... 395/82
5,355,304 10/1994 DeMoranville et al. .......... 364/413.02
5,355,439 10/1994 Bernstein et al. ........................ 395/82
FOREIGN PATENT DOCUMENTS
5514157 1/1980 Japan ............................... IB23K 9/12
4/1978 U.S.S.R. ............................ B25J 5/00
2/1985 United Kingdom
... B25J 9/00
WO 87/06008 10/1987 WIPO ............................ G01N 35/00
OTHER PUBLICATIONS
[75] Inventors: Steven A. Bernstein, Los Olivos; Page
A. Erickson; Stephen Barker, both of
5,696,887
Dec. 9, 1997
G. Owens and R. Eckstein, “Robotic Sample Preparation
Station,” Analytical Chemistry, vol. 54, No. 13, pp
2347–2351, Nov. 1982.
S. Vere, “Planning in Time: Windows and Durations for
Activities and Goals.” IEEE Tran, on Pattern Analysis and
Machine Intelligence, vol. PAMI-5, No. 3, pp. 246—267,
May 1983.
(List continued on next page.)
Primary Examiner—Robert W. Downs
Attorney, Agent, or Firm—McDonnell Boehnen Hulbert &
Berghoff
[57]
ABSTRACT
A system for performing a plurality of independent analysis
procedures simultaneously, each the procedure having a
sample and at least one process step for operating on that
sample the system having a robotic device for moving
sample among a plurality of processing stations where the
robotic device comprises substrate to which are coupled a
plurality of tiles each one of the tiles disposed art a selected
location on the substrate; a plurality of reagent trays coupled
to the tiles each one of the reagent trays having substantially
identical shape and size; a robotic arm disposed for reaching
substantially all the tiles; and comprising means for coupling
and decoupling from a sample holder, the sample holder
having a means for holding at least one sample in a selected
position, whereby the robotic arm is capable of moving the
sample from a first one of the reagent trays to a second one
of the reagent trays; and a processor for selecting, at a
plurality of times a sample to be moved, and for directing the
robotic arm to move the sample to be moved; the processor
having means for directing the robotic arm to interleave the
process steps of the plurality of independent analysis pro
cedures in a sequence conforming to a predetermined range
of durations for each one of the process steps.
16 Claims, 18 Drawing Sheets
5,696,887
Page 2
OTHER PUBLICATIONS
J. Lindsey, et al., “Robotic workstation for microscale
synthetic chemistry: On-line absorption spectroscopy, quan
titative automated thin-layer chromatography, and multiple
reactions in parallel,” Rev. Sci. Instrum. 59(6), pp.
940–950, Jun. 1988.
G. Gordon, et al., “ORCA: optimized robot for chemical
analysis.” Hewlett-Packard Journal, vol. 44, No. 3, p. 6(14),
Jun. 1993.
Hamacher, “Basic Structure of Computers, Computer
Organization, McGraw-Hill, Inc., 1984, pp. 1–6.
Stelzner, et al.; “The Simkit System: Knowledge–Based
Simulaton and Modeling Tools in KEE.” IntelliCorp, Inc.,
1987, pp. 1–22.
Sjolund, et al., “Robot Task Planning: Programming Using
Interactive Computer Graphics.” Proc. 13th ISIR, 1983,
7–122+7–135.
Kawobe, et al., “Robot Task Planning System Based on
Product Modeling.” IEEE 1985 Compint – Computer Aided
Technologies, Sep. 1985, pp. 471–476.
Okino, et al., “Robot Stimulator in TIPS/Geometric Simu
lator,” Robotics & Computer Integrated Manufacturing vol.
3, No. 4, 1987, pp. 429–437.
Isenhour, “Robotics in the Laboratory.” Journal of Chemical
Information and Computer Sciences, 1985, pp. 292–295.
Isenhour, et al.; “ICRTS: An Expert System for Temporal
Optimization of Robotic Procedures,” J. Chem. Inf. Comput.
Sci. 1988, pp. 215–221.
Hayes, et al.; “A Guide to GUIs.” Byte, Jul. 1989, pp.
250–257.
U.S. Patent
Dec. 9, 1997
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F|G.3C-l
9.
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F|G.3C-3C
E
F|G.3C–3f
FIG3C-3g
FIG3C-3h
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FIG 3C-3i
FIG3C-3k FIG,3C-3
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O
F|G.3C-3r
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FIG 3C-37
FIG.3C-3y
U.S. Patent
Dec. 9, 1997
Sheet 12 of 18
5,696,887
FILL TRAY
WITH REAGENT
581
SEAL COVER
ONTO TRAY
TRANSPORT
TRAY TO
ROBOTIC DEVICE
PLACE TILES
ON ROBOT
684
PLACE TRAYS
INTO TILES
REMOVE
COVERS
FROM TRAYS
686
U.S. Patent
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Sheet 16 of 18
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5,696,887
2
ments. Very limited dye staining can be done on fresh or
recently living tissue without resorting to chemical process
ing. Typically a sample of tissue 2.0 to 2.5 square centime
1
AUTOMATED TISSUE ASSAY USING
STANDARDIZED CHEMICALS AND
PACKAGES
ters in area and 3 to 4 millimeters thick is utilized. The tissue
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of application
Ser. No. 07/740,285 filed Aug. 5, 1991, now U.S. Pat. No.
5,355.439, and application Ser. No. 08/218,143, filed Mar.
24, 1994, both filed in the name of inventors Steven A.
Bernstein and Page A. Erickson, titled “Method and Appa
ratus for Automated Tissue Assay”, and assigned to the same
10
assignee.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods and apparatus useful in
automated analysis or testing of tissue samples, and to
automated tissue assay using standardized chemicals and
packages.
2. Description of Related Art
The analysis of tissue is a valuable diagnostic tool used by
the pathologist to diagnose many illnesses and by the
15
In order to obtain information from a tissue sample it is
usually necessary to perform a number of preliminary opera
tions to prepare the sample for analysis. There are many
variations of the procedures to prepare tissue samples for
testing. These variations may be considered refinements to
adapt the process for individual tissues or because a par
ticular technique is better suited to identify a specific chemi
cal substance or enzyme within the tissue sample. However
the basic preparation techniques are essentially the same.
Typically such operations might include the processing of
the tissue by fixation, dehydration, infiltration and embed
ding; mounting of the tissue on a slide and then staining the
sample; labeling of the tissue through the detection of
various constituents; grid staining of tissue sections for
analysis by an electron microscope or the growing of sample
20
25
35
cells in culture dishes.
Depending on the analysis or testing to be done, a sample
may have to undergo a number of preliminary steps or
treatments or procedures before it is ready to be analyzed for
its informational content. Typically the procedures are com
plex and time consuming, involving many tightly sequenced
steps often utilizing expensive and toxic materials.
These procedures must usually be performed in a critical
order for each sample and each treatment is frequently time
dependent. Additionally the laboratory is often under
extreme pressure to perform many different analysis as soon
as possible, entailing many different procedures and tests.
A sample of tissue may undergo an optical microscopic
examination so that the relationship of various cells to each
other may be determined or abnormalities may be uncov
ered. The tissue sample must be an extremely thin strip of
tissue so that light may be transmitted therethrough. The
average thickness of the tissue sample or slice (often
referred to as sections) is on the order of 2 to 8 microns. A
relatively soft and pliable tissue such as might come from an
organ of the human body, in its fresh state cannot be
accurately cut into such thin sections. In addition, in order to
nucleus, the nucleolus, the cytoplasm and the cell
dyes to produce a contrasting appearance between the ele
To prepare good samples for microscopic examination the
initial step should kill the enzymic processes of the tissue
and should alter or denature the proteins of the cell through
fixation. The period of fixation may take several hours or
even a few days depending upon the tissue type, sample size
and type of fixative being used.
After fixation, the tissue sample is often dehydrated by the
removal of water from the sample through the use of
increasing strengths of alcohol or of some other dehydrating
fluid. Gradual dehydration is preferred because it causes less
distortion to the sample than a rapid dehydration process.
The alcohol is then replaced by a chemical which mixes
with wax or some other plastic substance which can perme
ate the tissue sample and give it a consistency suitable for
the preparation of thin sections without disintegration or
splitting. Fat solvents, such as chloroform or toluene are
commonly used for this step. The sample, which has been
dehydrated by the infiltration of alcohol, is next exposed to
several changes of solvent over a period that may last from
a few hours to days until the alcohol is completely replaced
by the solvent. The sample is then exposed to a wax which
is soluble in the solvent. If a paraffin type wax is used the
infiltration is at a temperature above its melting point. After
the wax infiltration the sample is allowed to cool and the
wax solidify so that the sample is entirely embedded in and
infiltrated by the wax.
A microtome is then utilized to cut thin slices from the
45
50
tissue sample. The slices are on the order of 5 to 6 microns
thick. The cut thin sections are floated on water to spread or
flatten the section. The section is then disposed on a glass
slide, usually measuring about 8 by 2.5 millimeters.
The wax is then removed by exposing the sample to a
solvent, the solvent removed by alcohol, and the alcohol
removed by decreasing the alcoholic concentrations until
eventually the tissue is once more infiltrated by water. The
infiltration of the sample by water permits the staining of the
cell constituents by water soluble dyes.
55
Prior to the development of automated procedures for the
preparation of tissue samples, it often took from 2 to 10 days
before the tissue could be examined under a microscope. In
more recent years automated processes have been developed
utilizing apparatus to transfer the sample from one fluid to
another at defined intervals, and as a result the preparation
time has been significantly reduced to between about 4 and
16 hours.
Variations in the materials used in the preparation of the
sample are advantageous under some circumstances. The
see the individual constituents of the cells, such as the
membrane, it is preferable to have them colored by different
ethanol, picric acid or mercuric chloride usually with for
malin. It should be remembered that in dealing with these
substances the containers holding the materials must be
suitable. For example mercuric chloride severely corrodes
metals and therefore should normally be contained in a glass
vessel.
medical researcher to obtain information about a cell struc
ture.
sample is then fixed in a material (a fixative) which not only
preserves the cellular structure but also stops any further
enzymic action which could result in the putrification or
autolysis of the tissue. While many substances can function
as a fixative, a 4% formaldehyde or a 10% formalin solution
is very common. Other common fixatives would include
65
use of ester wax allows sections 1 to 3 microns thick to be
cut with less contraction than that which occurs when
paraffin used. The sample is exposed to higher temperatures
5,696,887
3
when paraffin wax is used. The use of cellulose nitrate
embedding shrinks tissues less than wax, produces good
cohesion between tissue layers and permits large undistorted
sections to be cut 25 to 30 microns thick, if so desired. It is
clear that persons with skill in the art of tissue preparation
may use many different materials to which the samples may
be exposed.
Tissue staining is a procedure which is utilized to make
microscopic structures more visible. Perhaps the most com
mon stain materials are hematoxylin and eosin. Hematoxylin
is utilized to clearly stain the nuclei of cells dark blue. Eosin
is used to stains the cell cytoplasm various shades of red or
yellow, presenting a clear contrast to the blue stain of the
4
Sternberger, L.A. Immunocytochemistry (2nd ed. 1979).
John Wiley & Sons: New York. ISBN 0-471-03386-3.
Many pathology laboratories have in fact automated many
of the simple and routine procedures described above such
as simple staining or sample embedding. Where the same
procedure is repeated with great frequency, laboratories
10
nuclei.
Many synthetic dyes are derived from benzene which is
colorless but by changing its chemical configuration color
compounds are produced which are called chromophores. It
is these chromophores which constitute the bulk of the
different coloring dyes used in research and routine histol
Ogy.
There are many techniques by which sample tissues may
be stained and most of these techniques require exposing the
sample to various solutions. Histochemistry is the science by
which chemical reactions are used to identify particular
substances in tissues. In addition, many enzymes can be
detected by exposing a sample to a particular chemical
substance on which the enzyme is known to have an effect
such as turning the substance into a colored marker. Thus
from the above it can be seen that a sample tissue may be
exposed to various antibodies, enzyme labeled detection
systems, colormetric substrates, counterstains, washing
buffers and organic reagents.
Many experimental and observational research projects
involve experimentation to authenticate new techniques and
these experiments can be very extensive and time consum
Ing.
In addition to the techniques that prepare samples for
optical microscopy, techniques often must be utilized which
make the use of electron microscopes suitable in the exami
nation of tissue samples. Actually it has been found that the
pathological examination of almost any disorder makes
electron microscopy highly desirable and often essential.
Tissue samples for use with an electron microscope may
be fixed in glutaraldehyde or osmium tetroxide rather than in
the standard fixatives used for optical microscopy samples.
Usually very small samples of tissue are embedded in
methacrylate or epoxy resin and thin sections are cut (about
0.06 microns thick). Staining is most often done by colored
solutions and not dyes, and heavy metal salts are utilized to
enhance contrasts of density.
From the above brief description of some of the tech
niques and materials used by a pathologist in the examina
15
20
25
35
45
50
55
Alan R. Liss, Inc.: New York. ISBN 0-8451–4213-5.
London. ISBN 0-407-72903-8.
sequentially and thus subsequent samples may be subject to
a significant time delay.
Research laboratories often are required to perform non
routine analysis requiring many different test procedures. As
a result of this lack of repetitive procedures, research labo
ratories have relatively little automated equipment to assist
this lack of automation is that the equipment presently
available is dedicated to a limited number of procedures
most commonly performed. The equipment is not flexible
enough to permit a wide variety of operations to be easily
accomplished nor does the present equipment permit easy
and facile changes to the operations.
Another problem that has arisen in the art of repeated
testing is that of reagent supply. Typically, devices to per
form repeated testing must be loaded with bulk reagents, and
those bulk reagents must have sufficient volume that a
specimen slide can be immersed in the reagent, at least to the
level of the specimen. This can be wasteful of expensive
reagents. It can also result in substantial contamination with
the reagent of the back or sides of the slide, resulting in
significant carryover of the reagent and its chemical effect
into a next step, and a possible safety hazard for the operator
or support personnel.
Another problem that has arisen in the art of repeated
testing is that of packaging of reagents for tests. Typically,
devices to perform repeated testing comprise isolator pads,
essentially hydrophobic surfaces of glass or plastic, with
roughened areas to contain the reagent and smooth areas to
repel it. This can cause two problems. First, if too much of
the reagent is doled out by the operator, it can overflow the
isolator pad and mix with another reagent. Second, the
reagent has a near maximal surface/volume ratio, often
resulting in significant evaporation of the reagent before use.
SUMMARY OF THE INVENTION
The invention provides a system which performs a plu
rality of independent analysis procedures simultaneously,
possibly involving differing types of tissues and differing
process steps. The system comprises a robotic arm, which
may move the different tissue samples among a plurality of
processing stations, and a processor, which may select the
next tissue sample to move, when to move it, and where to
move it to. In a preferred embodiment, the processor may
direct the robotic arm to interleave the differing process
steps, for example by time division multiplexing.
In a preferred embodiment, the processing stations may
be disposed in a set of grid locations, so that the location of
any one processing station may be specified by an X
Childs, G. W. Immunocytochemical Technology (1986).
Culling, C. F. A., R. T. Allison and W. T. Barr. Cellular
Pathology Technique (4th ed. 1985). Butterworths:
samples simultaneously to the same testing procedure, i.e.,
parallel testing or through the use of multiple machines the
same result of parallel testing, is achieved. Alternatively the
laboratory may perform the same test repetitively, i.e.,
the researchers in their task. The most obvious reason for
tion of tissues, it can be seen that for a research laboratory
to carry out such a wide variety of processes and numerous
different tests assisting apparatus would be desirable and
almost mandatory. Other and further information about
tissue analysis and tissue assays may be found in the
following references, each of which is hereby incorporated
by reference as if fully set forth herein:
Bancroft, J. D. and A. Stevens. Theory and Practice of
Histological Techniques (3rd ed. 1990). Churchill
Livingstone: Edinburgh. ISBN 0-443-03559-8.
have often designed specialized machines to perform the
often repeated testing simultaneously on many samples.
Typical of such machines are the equipment used in the
routine analysis of blood samples. The equipment used in
this type of laboratory is capable of treating multiple
65
coordinate and a Y coordinate, and possibly a Z coordinate
for height. The robotic device may comprise a bench robot
with sufficient degrees of freedom that it is able to reach each
5,696,887
7
8
could be immersed to a predetermined depth and retained in
the solution tray for a precise time. It should be clear that
each grid location may have a solution tray having different
depths or different dimensions. Alternatively, a grid location
could contain a slide holder or other peripheral equipment
capable of performing a single function on the sample.
The robotic equipment or robotic arm may be controlled
by a standard PC computer. The assay development software
is graphic in nature and places a model of the peripheral grid
on the screen of the computer. While each tissue assay may
have all its steps preprogrammed the assay development
software permits the steps of the procedure or the timing of
the steps to be altered. The graphic nature of the presentation
permits laboratory personnel to alter such elements without
the necessity of relying on a computer or programming
10. For example each work module 13 may include a floppy
disk which would contain the physical characteristics of the
work module, such as its height, width and length. The
expert.
The process control software associated with the PC may
monitor the progress of the assays, may permit manual
override of an automatic operation, and most importantly,
may permit scheduling of multiple assays simultaneously in
parallel through the use of time interleaving of the various
steps in the test procedures. Thus while sample #1 may be
disposed at a workstation in a grid location where it under
goes a drying operation, sample #2 may be located in a tray
containing a staining solution while sample #3 is undergoing
a fixation step. The timing of each step is accurate and the
system interleaves the steps and utilizes the “waiting” or
processing time between steps in a single procedure to
perform operational steps on other samples which may be
undergoing completely different preparation.
customized data for each module would be fed into the
10
15
25
device 10 and the laboratory bench top 11. A template may
be used to represent the operational area and to assist in
defining the exact location of each workstation.
Located on the bench top 11 are one or more work
35
45
modules 13. A control station 14 is located adjacent to the
laboratory bench 11. The control station 14 may include a
typical PC type computer 15, such as an IBM-compatible
computer having an Intel x86 processor, or a computer
similar thereto, mounted on a desk 16 or other working
surface. It would be clear to one of ordinary skill in the art,
after perusal of the specification, drawings and claims
herein, that other types of computers may be utilized to
control the movement of the robotic arm 10. A printer 17 is
shown although other peripheral equipments may be utilized
in conjunction with the computer 15.
Referring to the bench top 11, a plurality of locating holes
12 are disposed at predetermined fixed locations relative to
the robotic device 10. The locating holes are designed to
receive modular workstations 13. Each modular workstation
13 is designed to be used in the performance of a particular
process or step in one laboratory task or test procedure. Thus
each function required to be performed in a task is associated
with a work module 13 which has a predisposed known
position on the work bench 11.
There are a number of methods by which the location of
a particular work module 13 can be supplied to the computer
In a preferred embodiment, the robotic device 10 is
capable of travel in an X direction along a first cable driven
bearing 20 (actuated by a first cable drive 20a). Disposed at
right angle to and vertical with respect to the first cable
driven bearing 20 is a second cable driven bearing 21
(actuated by a second cable drive 21a), capable of traversing
the first cable drive 20a. Coupled to the cable drive 21a is
a third cable driven bearing 22 (actuated by a third cable
drive 22a) disposed at a right angle. A robotic hand 23 is
mounted on cable drive 22 and comprises a spring loaded
solenoid 23a coupled to a rubber securing ring 23b. The
securing ring 23b is capable of coupling to a sample carrier
23c. The sample to be assayed (which may be a tissue
sample) is mounted on the sample carrier 23c.
Thus the hand 23 on which the sample is mounted is
capable of X movement along cable driven bearing 20, Y
movement along cable driven bearing 21, and Z movement
along cable driven bearing 22. The system illustrated is thus
capable of motion relative to three axes. Although the
system is illustrated using cable driven bearings 20, 21 and
22, it would be clear to those skilled in the art, after perusal
of this application, that other robotic equipment could be
LABORATORY BENCH AND ROBOTIC
DEVICE
FIG. 1 shows a robotic device for use with the invention.
FIG. 2 shows a laboratory setup having robotic equipment
like that shown in FIG. 1. The equipment may include a
robotic device 10 mounted on a standard laboratory bench
top11. The bench top 11 defines the operational area reach
able by the robotic device 10. The bench top 11 may have
integral therewith a plurality of locating elements such as
holes 12. Alternatively, the locating elements may be dis
posed on a separate base disposed between the robotic
central processing unit of the computer and would query the
operator, for example through a CRT display, to provide the
location of the work module. The operator through the
keyboard input would specify the location of the module on
the locating grid. Thus for each work module or step of a
task the computer would have stored in its memory the
physical characteristics and location of the module.
provided that could decrease or increase the number of axes,
that other techniques other than cable drives and cable
driven bearings, (such as lead screws, gears, belts, or other
devices) could be used, that such other equipment or tech
niques would be workable, and are within the scope and
spirit of the invention.
Typically, the range of movement along the X axis may be
about 76 inches, along the Y axis about 19 inches, and along
the Z axis about 18 inches. Such a typical range of move
ment could provide approximately 15 cubic feet of opera
tional area.
SYSTEM OPERATION
50
55
65
In order to illustrate the operation of this invention, let it
be assumed that the laboratory has five example tasks to
accomplish, each having five example steps. For purposes of
illustration, the five steps in each of the five tasks will be
utilized to demonstrate the multitasking capabilities of the
invention. The five tasks and the five steps of each of the
tasks are shown in Table 1 herein.
It is apparent from Table 1 that some of the tasks utilize
the same steps such as Pad 1 or Buffer 1. If these steps were
to be carried out in accordance with the principles of this
invention, it would be necessary to provide only 14 work
modules even though 25 steps were being performed. Dis
posed on the grid would be a separate work module for each
of the 14 different steps listed above. Thus there would be a
Pad 1 module to be used in carrying out seven of the above
steps. Alternatively, the user could provide multiple
modules, each capable of performing the pad function. A
Buffer 1 module would be used for
5,696,887
9
10
TABLE 1
and for grasping. The hand retaining the grasped sample
would move the sample to the location of the work module
for Task #1, Step #1, i.e., Buffer 1. The sample would be
Five Tasks
Task #1
Step #1
Step #2
Step #3
Step #4
Step #5
Task #2
Step #1
Step #2
Step #3
Step #4
Step #5
Task #3
Step #1
Step #2
Step #3
Step #4
Step #5
Task #4
Step #1
Step #2
Step #3
Step #4
Step #5
Task #5
Step #1
Step #2
Step #3
Step #4
Step #5
freed from the hand and left at the work module. The hand
would proceed to the location of sample #2 where it would
Basic Fuchsin Staining
grasp the sample and carry it to the work station where Task
Buffer 1
Buffer 2
IBasic Fuchsin
Pad 1
Buffer 2
Azure II & Methylene Blue Counterstaining
#2, Step #1 would be performed.
10
Azure II
Pad 1
Buffer i
Pad 1
sample. It should be noted that the design of the Buffer and
Pad work modules permit the simultaneous treatment of at
least two samples from different tasks. Alternatively, two
work modules could be provided so that each sample could
Methylene Blue
Tissue Fixation
Isotonic Rinse
15
Buffer 1
Buffer 2
Buffer I
Pad 1
20
Blocking Antibody
Pad 1
Buffer 1
Slide Silinizing
APTES
Toluene
25
Water
Pad 1
Oven
30
five of the steps and a Buffer 2 module for two of the steps.
Each of the remaining steps would have a module disposed
on the grid to perform the necessary work associated with
the step.
It is often essential that the step of the task be performed
within certain time limits. The timing of some steps can be 35
critical. FIG. 4 is a flowchart showing a time line for the five
steps of the tasks in Table 1. It should be noted that Task #1,
Step #1 commences at 9:00 and has a duration of approxi
mately 15 minutes, inclusive of the time necessary to
transport the sample to the location where Step #2 is
performed. Thus Step #2 will commence at approximately
9:15. It should be noted that the timing for the start of Step
#2 has some leeway in that it can commence between 9:15
and 9:18, providing leeway of three minutes. Step #2 has a
duration of approximately 11 minutes and the sample is 45
transported to the location where Step #3 will be performed.
The time for performing Step #3 is critical as indicated by
the lack of interval for the starting times. Step #3 must
commence at 9:26. Fourteen minutes later the sample is
undergoing Step #4, which can commence any time between 50
9:40 and 9:50. The last Step #5 is performed at 9:51. It
should be noted that if each Step is commenced at the outer
time limit Step #5 may not begin until 10:22.
In a similar manner it can be determined from FIG. 4 that
the five steps of Task #2 may consume 1 hour 34 minutes,
and gasps the sample #5 and transports it to the module for
Task #5, Step #2. Following the path illustrated in FIG. 5,
the hand proceeds from the Task #5, Step #2 module to Task
#3, Step #3 module where it grasps sample #3 and transports
it to Task #3, Step #2 module where the sample is deposited.
The hand then returns to the location of the first sample
which is in the module associated with Task #1. Step #1 and
takes it to the module for Task #1, Step #2. The hand returns
to the location sample #4 and carries it to Task #4, Step #2
and then at the appropriate time transports the same sample
to Step #3 of Task #4.
At this point in the operation of the system, the computer
detects that Task #1, Step #3 and Task #2, Step #2 are both
scheduled to start at the same time, 9:26. In order to resolve
the conflict the system utilizes a technique, herein termed
“fuzzy timing”, to process the control of the robotic hand
and optimize the process. Fuzzy timing may comprise the
window of time during which each process (Task) step may
occur without affecting the process results. Some steps of a
process may be critically timed, i.e., the time required for
that step is exact, such as Task #1, Step #3 in FIG. 5, but in
general most steps a process the timing is less critical and
may comprise any amount of time within a known range and
thus are noncritical in their timing, such as Task #2, Step #2,
which has a window of four minutes, as shown in FIG. 5.
The system of this invention uses these windows of time to
advantage as to optimize (minimize) the time necessary to
complete the multiple tasks.
The use and advantages of “fuzzy timing” can be illus
trated by considering two different tasks, each having a
process step terminating at the same time or within moments
of the another. Assuming that both steps are critically timed
in so far as the termination time is concerned, it is apparent
that both samples from the two different steps can not be
moved to the next step in each process simultaneously since
concurrent movement of two samples is not within the
capabilities of this embodiment. Thus it is necessary to
55
adjust the starting times for the two steps relative to each
other so that the ending times will allow for the movement
of each sample to its next process step. While this can be
done quite easily, it is clear that the mere adjustment of a
starting time for a step in the process may well cause other
timing conflicts. It is possible that under such conditions the
system could not support simultaneous throughput of mul
tiple processes unless the timing was altered.
Fuzzy timing allows the system additional flexibility
65
timed process step, conflicts will be minimized through the
adjustment of timing at the step level. rather than by shifting
the timing of the whole process or task.
Task #3, 1 hour 9 minutes, Task #4, 1 hour 17 minutes, and
Task #5, 1 hour 16 minutes. Thus if the five steps of the tasks
shown were to be performed sequentially the total time to
completion would be 6 hours 38 minutes.
Referring to FIG. 5, the multitasking method of this
invention is therein illustrated to show the time interleaving
of the steps of the multiple tasks. Assuming again for
purposes of illustration and simplification of explanation
that we are desirous of performing the same five steps for the
same five tasks. Under the control of the computer the
robotic hand would be commanded to obtain sample #1 or
alternatively the sample could be brought to the robotic hand
be treated in a different module.
After locating sample #5 in the Task #5, Step #1 module,
the robotic hand returns to the module for Task #5, Step #1
Primary Fixative
Secondary Fixative
Immunocytochemistry
Each of the five samples would in turn be grasped by the
robotic hand and transported to the work module associated
with the first step of the task to be performed on each
since by providing a window of time at each noncritically
5,696,887
12
402 is disposed next to a second stub 323 in its pair, the
second stub 323 being the matching stub 323 aligned with
11
STANDARDIZED CHEMICALS AND
PACKAGES
the first stub 323.
FIGS. 3A-1, 3A-2, 3A-3, 3A-4, and 3A-5 shows a stan
Each subdepression 323 comprises a pair of receiving
dardized tile for coupling to the robotic device.
As described herein, the robotic device 10 may be
mounted on a bench top 11 having a plurality of locating
elements such as holes 12 and having a plurality of work
modules 13 disposed thereon.
In a preferred embodiment, each work module 13 com
prises one or more tiles 301, each tile 301 comprising a
molded plastic piece having a top face 302 and a bottom face
10
holes 325.
303.
The bottom face 303 of the tile 301 comprises a relatively
flat plastic surface 304. possibly having one or more bottom
indentations 305 and bottom ribs 306, and having a set of
receiving wells 307 for insertion of a corresponding set of
15
fasteners 308. As shown in FIG. 3A-4, the fasteners 308 fit
through a set of holes 12 for a designated location on the
bench top 11, and are coupled to the receiving wells 307 for
fastening the tile 301 to the top surface of the bench top 11.
In a preferred embodiment, the fasteners 308 comprise
screws, but those skilled in the art will recognize, after
perusal of this application, that other types of fasteners
would also be workable with the devices and substances
20
25
described herein, and are within the scope and spirit of the
invention.
The top face 302 of the tile 301 comprises a set of
receiving areas 309 for insertion of a corresponding set of
standardized packages 401. The top face 302 also comprises
a set of one or more top indentations 310 and top ribs 311.
A set of holes 312 are disposed in at least some of the top
indentations 310, so that liquids in those top indentations
310 may drain. Each receiving area 309 comprises a depres
sion 320, into which a package 401 (FIG. 3B-5) may be
placed.
Each depression 320 comprises a pair of side walls 321
disposed parallel to each other, a pair of intermediate bar
riers 322 disposed so as to divide the depression 320 into a
set of three subdepressions 323, each intermediate barrier
322 having a pair of stubs 324. Each pair of stubs 324 is
aligned with each other and disposed parallel to the side
walls 321, so that a package 401 may be snugly fitted into
one of the three subdepressions 323.
Each stub 324 comprises a first and second stub side 325
When a package 401 is fitted into a side one of the three
subdepressions 323, it is disposed with a first package side
402 (FIG.3B) disposed next to a first side wall 321 and with
a second package side 402 disposed next to one of the stubs
323, in particular, next to one of the stub sides 324. A first
end of the second package side 402 is disposed next to a first
35
45
The tray 406 comprises a tray frame 420, having a
rectilinear shape with a top surface 421. The top surface
includes the tray surface 413 for bonding with the cover 407,
and also includes a handle region 422 with a hole 423
disposed therein.
The cover 407 also comprises a cover lip 414 disposed on
at least one end of the package 401, having a sufficient size
to be grasped by an operator and removed from the tray 406.
The tray frame 420 comprises a pair of side surfaces 424,
disposed perpendicular to the top surface 421. The side
surfaces 424 form the package sides 402 and the ends 403
of the packet sides 402.
The tray frame 420 comprises a first end surface 425,
disposed perpendicular to the top surface 421 and to the side
surfaces 424, and forming a box shape underneath the
50
handle region 422 and the hole 423, providing additional
sturdiness in that region.
The tray frame 420 comprises a second end surface 426,
disposed perpendicular to the top surface 421 and to the side
surfaces 424, and having the spring lock 405 disposed
thereon.
55
The tray frame 420 comprises a set of tray ribs 427,
disposed underneath the top surface 421 and near the side
surfaces 424, providing additional sturdiness to the tray 406
and the side surfaces 424.
stub 323, while a second end of the second package side 402
is disposed next to a second stub 323, the second stub 323
The tray frame 420 is coupled to a well frame 440, which
comprises a rectilinear shape having a pair of well sides 441,
being the matching stub 323 aligned with the first stub 323.
Alternatively, a package 401 may be fitted into a center
one of the three subdepressions 324. In this case, it is
disposed with a first package side 402 disposed next to a first
pair of stubs 323, and a second package side 402 next to a
second pair of stubs 323. A first end 403 (FIG. 3B-2) of the
second package side 402 is disposed next to a first stub 323
in its pair, while a second end 403 of the second package side
FIG. 3B (comprising 8 parts, individually FIGS. 3B-1,
3B-2, 3B-3, 3B-4a, 3B-4b, 3B-3c, 3B-5, and 3B-6) shows a
standardized package for coupling to the tile.
In a preferred embodiment, a standardized package 401
comprises a molded plastic tray 406 and a thin cover 407
affixed to the tray 406, such as by a heat weld, a glue, or
other known means. In a preferred embodiment, the thin
cover 407 may comprise a plastic or metallic sheet 408,
laminated on an outside side 409 with plastic and printed
thereon with identifying information, and coated along an
edge area 410 on an inside side 411 with a fixative 412 and
affixed by means of that edge area 410 to a corresponding
tray surface 413.
In a preferred embodiment, the fixative 412 comprises a
heat weld, but those skilled in the art will recognize, after
perusal of this application, that other types of bonding
techniques would also be workable, such as crimping or
welding, or glue, and are within the scope and spirit of the
invention.
and a stub end 326. The stub sides 325 for the stub 324 are
disposed parallel to the stub sides 325 of the matching stub
324, and parallel to the side walls 321. The stub end 326 for
the stub 324 is generally disposed so that the stub 324 is
relatively short compared with the package 401.
holes 325 for insertion of a corresponding lever 404 (FIG.
3B-4C) and a corresponding spring lock 405 (FIG.3B-6) of
the package 401 to be disposed in the subdepression 323.
When the package 401 is fitted into the subdepression 323,
the lever 404 of the package 401 is disposed in a first one of
the receiving holes 325, and the spring lock 405 of the
package 401 is disposed in the second one of the receiving
a well bottom 442, a set of wells 460, a first well end 443
near the first end surface 425, and a second well end 444
near the second end surface 426.
The wells 460 each comprises a truncated wedge shape,
65
having a single well bottom 461 that is U-shaped, with the
plane of the U-shape parallel to the side surfaces 424, and a
pair of single well sides 462 that are flat and each have a
5,696,887
13
trapezoidal shape. Each single well bottom 461 comprises a
set of three relatively straight surfaces, a well horizontal
bottom 463 that is relatively flat and horizontal (and may
comprise a V shape with a arms of the V shape disposed
about 2.5 degrees from horizontal), and a pair of well
semibottoms 464 that are flat and disposed at an angle of
about 9.5 degrees from the vertical.
5
The single well bottoms 461 are disposed in a continuous
sequence so as to merge to form the well bottom 442. The
well bottom 442 is therefore formed without seams and with
ridges 465 formed by well semibottoms 464 adjacent to each
10
by orienting a slide 540 with a specimen 541 vertically for
insertion into the inside well 521, i.e., with the flat surfaces
15
thereon.
A second well 460 with a label466 of “1” is disposed near
the second end surface 426 and has a set of end ribs 467
disposed thereon.
The lever 404 comprises a right-angled lever lip 480,
having a first lever surface 481 and a second lever surface
482, supported by a set of lever ribs 483 disposed between
This capillary action is particularly promoted if the slide
540 is coupled to a second slide 540 to form a slide pair 543,
with the specimen 542 sandwiched between the slide 540
and the second slide 540 of the slide pair 543, and with the
slide 540 and the second slide 540 maintained a selected
separation distance apart of preferably about 146 microns
25
30
35
45
50
is not so large that nonspecimen parts of slides 540 or slide
pairs 543 (such as the back or sides) are regularly exces
sively contaminated. This selected volume of liquid also has
the advantage, particularly when held in an inside well 521
having a single well bottom 461 with relatively steep sides
(formed by the well horizontal bottom 463 and the well
semibottoms 464), that there is a reduced surface/volume
ratio. This provides for lesser evaporation of the liquid in the
It has also been found by the inventors that the selected
shape of the inside well 521 is particularly advantageous.
This particular shape promotes self-levelling and reduced
evaporation, as noted herein. Moreover, this particular shape
promotes centering within the inside well 521 of small
amounts of liquid (about 150 microliters), due to surface
tension repulsion of the liquid by the well semibottoms 464.
Centering of the liquid promotes capillary action when a
slide 540 or slide pair 543 is inserted into the inside well
bottom 524 of the inside wells 521.
inside wells 521.
slide pairs 543 may be inserted into the inside well up to
about three times). However, this selected volume of liquid
inside well 521.
55
surfaces 424, and having a taper from thicker near a bottom
end 523 disposed near the bottom524 of the inside wells 521
to thinner near a top end 525 disposed farther from the
Each well divider 522 comprises a center 526, at a bottom
curve of the U-shape, that has an indentation 527, thus
forming two lips 528 disposed between each adjacent pair of
5,002,736; 5,023,187; and 5,116.727, and may be used in
is particularly advantageous. This selected volume of liquid
is generally sufficient to perform all the steps of typical
immunohistochemical stains and other assay protocols
(generally, with this selected volume of liquid, slides 540 or
ferred embodiment, the first lever surface 481 has at least
500 is disposed in parallel to the second end surface 426 and
sized to fit into the corresponding receiving hole 325.
An inside surface 520 of the tray 406 comprises a set of
inside wells 521 corresponding to the wells 460. Each
adjacent pair of inside wells 521 is separated by a well
divider 522. Each well divider 522 comprises a U-shaped,
with the plane of the U-shape perpendicular to the side
by reference as if fully set forth herein: U.S. Pat. Nos.
4,731,335; 4,777,020; 4.798.706; 4,801.431; 4.975,250;
conjunction with inventions therein.
It has been found by the inventors that the selection of the
particular volume, 750 microliters, for each inside well 521
underneath the second lever surface 482. The lever lip 480
is disposed at parallel to the first end surface 425 and sized
to fit into the corresponding receiving hole 325. In a pre
surface 501 comprises a section of the second end surface
426 having a pair of cuts 504 disposed thereon, a reinforced
spring base 505 disposed at a base of the pair of cuts 504,
and a pair of second spring ribs 506 disposed underneath the
second spring surface 426 near the cuts 504. The spring lip
+12 microns. However, those skilled in the art will
recognize, after perusal of this application, that slides of
differing sizes and selected separation distances would be
workable, and are within the scope and spirit of the inven
tion. For example, a selected separation distance for a slide
540 or a slide pair 543 for frozen tissue may comprise a
substantially larger size, such as about 200 microns. A
preferred embodiment of the slide pair 543 is shown in one
or more of the following U.S. Patents, hereby incorporated
the first lever surface 481 and the first well 460 and
one lip hole 484 disposed thereon, to promote mating at a
surface of the tile 301 near the receiving hole 325.
The spring lock 405 comprises a right-angled spring lip
500, having a first spring surface 501 and a second spring
surface 502, supported by a set of first spring ribs 503
underneath the second spring surface 502. The first spring
of the slide 540 being perpendicular to a plane of the ground.
When the slide 540 is inserted into the inside well 521, a
liquid content 542 of the inside well 521 will coat the
specimen 541 by means of capillary action.
Each well 460 is formed with a molded label 466 that is
unique within the package 410. In a preferred embodiment,
the labels 466 are formed by molding the plastic of the tray
406, but those skilled in the art will recognize, after perusal
of this application, that the labels could be workably formed
by alternative means, such as etching, printing, or scoring,
and that such alternative means are within the scope and
spirit of the invention. In a preferred embodiment, the labels
466 may each comprise a single digit “0”, “1”, “2”, “3", “4”,
“5”, “6”, “7”, “8”, or “9”. Alternatively, the number “10”
may be substituted for the digit “0”.
A first well 460 with a label 466 of “0” is disposed near
the first end surface 425 and has the lever 404 disposed
inside well 521 exceeds 750 microliters, the liquid will spill
over the bottom curve of the U-shape of the well divider 522.
and thus spill into the adjacent inside well 521.
In a preferred embodiment, the robotic device 10 operates
other.
The single well sides 462 are disposed in a continuous
sequence so as to merge to form the well sides 441. The well
sides are therefore formed without seams and without
ridges.
14
Each well divider 522 comprises a well top 527, at a pair
of top ends 528 of the U-shape, disposed with a gap 529
between the well top 527 and the cover 407.
In a preferred embodiment, the well dividers 522 are sized
so that each inside well 521 may hold 750 microliters (% of
a milliliter) of liquid without spilling over to an adjacent
inside well 521. However, if the amount of liquid in an
521.
65
Preferred filling amounts for content of the inside well
521 are about 350 microliters when the compound or
mixture is not too expensive, and about 200 microliters
5,696,887
17
detailed parts drawings for the first standardized slide carrier
18
The incubation oven 620 is triggered when first coupled
to the robotic system, and controlled to a temperature
selected by the control station 14. Typically, the control
560 and the second standardized slide carrier 600.
WORKSTATION DEVICES
In addition to packages 401, the tile 301 at a workstation
13 may be coupled to another type of device for operating
on samples, whether carried by slides 540, slide pairs 543,
or another carrying medium such as a beaker, test tube or
wafer. In a preferred embodiment, the tile 301 at a work
station 13 may be coupled to one or more of the following
station 14 will set the regulated temperature of the incuba
tion oven 620 to a room temperature such as 25 degrees
Celsius, will set the regulated temperature of the incubation
oven 620 to an operating temperature such as 95 degrees
Celsius a few minutes before the incubation oven 620 is to
10
devices:
The workstation 13 may comprise a centrifuge, a diffusion
device, a distillation device, or other separation device.
The workstation 13 may comprise a DNA crosslinking
device.
The workstation 13 may comprise an electroporator.
The workstation 13 may comprise a laser device or other
optical device.
The workstation 13 may comprise a microwave device, a
15
20
shielded radioactive sample, or other radiation source, such
as a source of electromagnetic or ionic radiation.
The workstation 13 may comprise an incubation oven or
other heating unit.
The workstation 13 may comprise a refrigeration element
or other cooling unit.
FIG. 3D (comprising 2 parts, individually FIGS. 3D-1 and
3D-2) shows a workstation having an incubation oven and a
carrying medium for inserting slides 540 or slide pairs 543.
In a preferred embodiment, an incubation oven 620 com
prises a chassis 621, an incubation chamber 622 a set of heat
25
630, and a set of heat fins 629.
The incubation chamber 622 is supported by the chassis
621 and comprises a set of chamber walls 631 disposed in
a generally rectilinear form 632 with a set of rounded
corners 633 to form a first part of a sealed fluid-tight box 634
when the carrying medium 630 is disposed for operation.
When the carrying medium 630 is disposed for operation,
the slides 540 or slide pairs 543 in the carrying medium 630
may be heated with moist heat formed by heating the
incubation chamber 622 while disposing a hydrating fluid
therein, and thus incubated. Incubation of slides 540 or slide
35
45
50
55
of the slide 540 or slide pair 543 to include the sample.
A horizontal plate isolates the heat exchanger fins 623
from the hydration fluid supply 624. The heat exchanger fins
623 may each comprise a resistive element such as a metallic
wire, coupled to a voltage source 634 disposed outside the
incubation chamber 622. The voltage source 634 is coupled
to a voltage regulator 635 to regulate the temperature of the
art.
ing and cooling without drying out the sample. Alternatively,
other assay protocols, such as those for heating a xylene
mixture, may require a relatively dry heat.
In a preferred embodiment, the hydrating fluid 636 may
comprise (per 10 liters) 9980 milliliters nanopure water, 20
milliliters Tween-20, and 10 grams sorbic acid. However,
those skilled in the art would recognize, after perusal of this
application that plain water, a known buffer solution, or
workable for the hydrating fluid 636, and that such sub
stances would be within the scope and spirit of the invention.
The internal cooling element 625 is disposed in the
chassis 621 near the incubation chamber 622 to cool the
incubation oven 620 and those of its elements that do not
need to have an raised temperature. The internal cooling
element 625 comprises a fan 638 coupled to the voltage
source 634 and to a temperature regulator 639, such as a
thermostat, to maintain the chassis 621 at a selected tem
perature. The heat fins 629 also serve to aid in regulating the
incubation chamber 622 to a selected temperature. Tempera
ture regulation is known in the art.
The fluid waste receiver 627 comprises a chamber for
receiving excess hydrating fluid 636 not evaporated by the
heat exchanger fins 623, and other fluids that may be
condensed by the internal cooling element 625. The fluid
waste receiver 627 may be detachable for emptying.
The receiving element 628 comprises a set of receiving
slots 640 molded into a bottom 641 of the incubation
chamber 622, disposed to receive a set of feet 641 of the
carrying medium 630. The carrying medium's feet 641 are
incubation chamber 622, and thus of the slides 540 or slide
pairs 543, to a selected temperature in steps of 1 degree
Celsius between ambient temperature to about 100 degrees
Celsius. Heating elements and regulators are known in the
The selected level of hydrating fluid 636 may be adjusted
to account for differing assay protocols. For example, an
another substance for incubation of tissue, would also be
The heat exchanger fins 623 are disposed in the incuba
exchanger fins 623 for each slide 540 or slide pair 543, or at
the least, for each pair of slides 540 or slide pairs 543. Each
one of the heat exchanger fins 623 has a height sufficient to
heat the entire slide 540 or slide pair 543, or at least a portion
selected level of hydrating fluid 636 is maintained. The
selected level of hydrating fluid 636 is maintained by means
of an automatic replenisher having a combination of a
reservoir and valve, disposed to maintain a constant level of
hydrating fluid 636 in the fluid well 637 available for
evaporation into the incubation chamber 622, similar to a
bird feeder. The fill/drain control 626 provides for filling and
draining the hydrating fluid 636 from the fluid well 637.
Flow regulation and fluid level regulation are known in the
assay protocol for hybridization may generally require heat
pairs 543 is known in the art.
tion chamber 622 in an array. The array is disposed to match,
but not contact, a set of slides 540 or slide pairs 543 disposed
in the carrying medium 630. There should be one of the heat
the incubation oven 620, and a fluid well 637 in which a
art.
30
exchanger fins 623, a hydration fluid supply 624, an internal
cooling element 625, a fill/drain control 626, a fluid waste
receiver 627, a receiving element 628 for a carrying medium
be used in a process step, and will set the regulated tem
perature of the incubation oven 620 to a room temperature
or to a second operating temperature such as 37 degrees
Celsius after the incubation oven 620 is used in a process
step and before it is to be used in a second process step. Each
process step designating the incubation oven 620 indicates
an operating temperature for that process step.
The hydration fluid supply 624 comprises a source, such
as a bottle, into which a hydrating fluid 636 is placed and
from which hydrating fluid 636 is drawn during operation of
similar to those of the first standardized slide carrier 560 or
65
the second standardized slide carrier 600, so the receiving
element 628 is similar to the top indentations 310 of the tile
301.
5,696,887
23
can be stored on a disk and the apparatus set up accom
plished by reading the information off a disk into the
memory of the computer.
In creating the template the operator uses a mouse to draw
replicas of each station on the screen, such as shown in FIG.
7, a template building screen. Each station is given a unique
identification which may be a name, symbol or code. The
dimensions of the station may be drawn on the screen and in
particular it is essential that the height of the work station is
recorded. The position, identification, height and other
dimensional criteria are stored in the RAM memory of the
computer CPU. When the template is completed it may be
stored to disk as a template file, to be recalled as needed.
As is not unusual in the operation of computers, provi
sions are made to add, delete, move, resize or duplicate any
of the stations. Any available template previously stored
may be recalled to be used or to assist in the creation of new
templates. Of course the apparatus may have the ability to
enable the operator to print out a graphic replica of the
screen and a list of station positions, identifications, heights
or other dimensions.
Once the template is complete the operator may use the
stations of the template to create a process, step by step.
The process builder, like the template builder, uses a
graphic replica of the workstation area on the computer
screen, such as shown in FIG. 8, a process building screen.
One of the templates previously created by the template tool
builder described above, is recalled from memory and
displayed on the screen together with the work area. The
screen cursor is moved to the desired station icon and the
particular station is selected. This procedure may utilize a
mouse and a point and click procedure.
Each station of the process is selected in sequence and the
station is then added to a list denoting the steps of the
process in sequential order. The robotic device would ulti
mately be controlled to move to each of these stations in the
order in which they were added the process list. Since the
characteristics of each work station were previously stored
in the computer, the robotic device would be programmed
for the proper movement. For example, the height of each
station was previously stored in the memory, and if the
robotic arm were to traverse the area in which a high work
station was located, it would be instructed to elevate the
hand so that any sample mounted thereon would clear the
high work station. It is also possible to design the opera
tional area to have clear paths or lanes defining travel routes
for the robotic device 10. In any event, the movement of the
robotic device among the workstations may be designed to
be free of collisions based upon recognition of the entity,
position and geometry of the work stations. As will appre
24
PSEUDO CODE FOR DESIGNING OR
RUNNING NEW PROCESSES
5
10
TABLE 2
set up screen;
20
draw robot replica graphic;
draw grid;
display mouse cursor;
select template design tool;
case (edit tool)
add:
draw new station on screen via mouse by
dragging mouse away from start point while
having mouse button 1 depressed;
update screen with a rectangle being
displayed along cursor displacement;
enter id via keyboard;
position height of station;
store position and id;
25
move cursor to station via mouse;
select:
click mouse to select;
selected station changes color to show it is
selected;
click mouse button 1 to delete;
delete:
Inove:
35
place move crosshair on selected station;
place cursor on crosshair;
press mouse button 1 down and drag station to
new position;
screen update after each new grid position
move;
place resize crosshair on selected
resize:
station;
place cursor on crosshair;
press mouse button 1 down and drag station to
new size;
screen update after each new size;
duplicate: get current selected station position, size
and height information;
offset duplicate to new position;
45
add id;
store new station position and id;
After the station sequence has been entered and the times
for each step recorded, the process may be stored to disk as
a process file. The process file may be loaded in the future
and the apparatus used to run the same process at a later date.
Of course the template file may be linked to the process file
55 SO
TABLE 3
Process Builder
procedure process tool();
herein, the minimum time may be specified to be zero, and
apparatus.
Template Builder
procedure template tool();
remain at the work station would be recorded. As noted
the maximum time may be specified to be infinity. The times
for each station, except where the timing is critical, would
allow the system a timing window which can be used to
avoid timing conflicts between different steps of separate
tasks and thus maximize the multitasking capabilities of the
of the specification, drawings and claims herein, that modi
fication of known processor systems to perform the func
tions disclosed in this pseudocode (as well as in other
pseudocode disclosed herein) would be a straightforward
task and would not require undue experimentation.
15
ciated as the number of work stations increase the amount of
information that should be considered in order to avoid
collisions and otherwise avoid conflicts in instructions also
increases.
Following the graphic design of the steps of the process,
the process list would be called up on the screen and the
procedure for each step would be imparted, such as shown
in FIG.9. This procedure would essentially indicate a range
of time each sample should remain at each station. For each
step a minimum time and a maximum time for the sample to
The method carried out by the control station 14 for
template building and process building may be described by
pseudocode shown in Tables 2–3 herein, respectively. It
would be clear to one of ordinary skill in the art, after perusal
set up screen;
draw robot replica graphic;
draw grid;
65
draw process list;
display mouse cursor;
case (file tool)
get template:
display list of template files;
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show five processes which are in progress, and may show for
each process the current step it is on, the total time it has
TABLE 3-continued
Process Builder
select via mouse cursor;
open selected template;
display template stations on screen;
hold station record in RAM;
display list of process files;
get process:
select via mouse cursor,
open selected process;
display process list in list window;
display associate template stations on
the screen;
save process:
hold process station records in RAM;
display list of process files;
select via cursor or enter new name via
keyboard;
15
store process file to disk;
case (file tool) end;
case (select tool):
if cursor in work station area and on a station and mouse
button 1 down then add station to process list;
if cursor in process list and on list member and mouse
button 1 down then delete from list;
case (select tool) end,
case (window select)
Process List:
Run/Control:
(1) set up screen;
(2) display process in list mode;
(3) enter min/max time via keyboard;
(4) scroll down screen;
(5) do steps 3–4 until finished;
(6) exit back to previous window;
25
return to Run/Control window;
end (process tool);
30
that when a process is called up from storage and run on the
computer the template files used in the process may be
automatically called up and displayed on the computer
Screen.
The procedure list on which the times at each step were
recorded may be called up at any time and for the stations
still not used by the robotic device, adjustments to the timing
35
a status section 64.
The display section 62 may show a representation of the
robotic device 10, bench top 11, holes 12, work modules 13,
and related equipment. For example, the display section 62
may show positions for workstations 13 for a selected
process.
45
50
55
The menu section 63 may show command options and
suboptions which are available to the operator and may
allow the operator to select one or more command options
and suboptions. For example, the menu section 63 may have
a menu with the command options “GET PROCESS”,
FIG. 9 shows a process timing screen 91 as viewed by an
operator. A process timing screen 91 may be shown on a
display device coupled to the computer 15, such as a display
monitor, in like manner as the multitask monitoring screen
61. The process timing screen 91 may comprise a plurality
of lines 92, each of which may have an identifier section 93.
a name/descriptor section 94, a minimum time section 95
and a maximum time section 96.
When using the process building tool, described herein,
the operator may view the process timing screen 91 and
enter minimum times (in the minimum time section 95) and
maximum times (in the maximum time section 96) for each
process step at each line 92. Each process step may thus have
name or descriptor in the name/descriptor section 94.
The minimum time section 95 for a line 92 may specify
a minimum time which the designated process step may
take, which might be zero. If the minimum time is zero,
additional data may be noted to indicate whether the desig
nated process step may take a single tick of a timing clock
for the robotic device 10, or if the designated process step
“BUILD PROCESS”, “PROCESS LIST", “GET TEM
The status section 64 may show a set of status information
about processes. For example, the status section 64 may
The workstation section 85 may show a set of names or
other identifiers of workstations 13. The operator may select
one or more workstations 13 for inclusion in a process, by
means of a pointing device, such as a mouse.
When using the process building tool, described herein,
the operator may view the process building screen 81 and
manipulate the commands and elements thereon by means of
a pointing device, such as a mouse. A detailed description of
how the operator may use the process builder tool is given
a line 92 with an identifier in the identifier section 93 and a
PLATE” and “BUILD TEMPLATE”. The operator may
display available command options and select one or more
command options in the menu section 63, by means of a
pointing device, such as a mouse, as is well known in the art.
FIG. 8 shows a process building screen 81 as viewed by
an operator. A process building screen 81 may be shown on
a display device coupled to the computer 15, such as a
display monitor, in like manner as the multitask monitoring
screen 61. The process building screen 71 may comprise a
display section 62, a menu section 63, and a status section
64, in like manner as the multitaskmonitoring screen 61, and
herein.
VISUAL OPERATOR INTERFACE
by an operator. A multitask monitoring screen 61 may be
shown on a display device coupled to the computer 15, such
as a display monitor. The multitask monitoring screen 61
may comprise a display section 62, a menu section 63, and
herein.
a workstation section 85.
could be made provided that the steps in the process which
are to have their timing altered have not been reached. Thus
the operator can adjust the timing of the steps even as the
process is running.
FIG. 6 shows a multitask monitoring screen 61 as viewed
taken (both for the current step and for the entire process).
and the time remaining that it will take (both for the current
step and for the entire process). Note that elapsed time for
the current step may be zero because the robotic device 11
might wait for the proper time before depositing the sample
in the workstation 13 for that process step, e.g., holding the
sample in the robotic hand 23 if travel from a prior step took
less time than expected. The status section 64 may also show
the X, Y and Z position of the robotic arm.
FIG. 7 shows a template building screen 71 as viewed by
an operator. A template building screen 71 may be shown on
a display device coupled to the computer 15, such as a
display monitor, in like manner as the multitask monitoring
screen 61. The template building screen 71 may comprise a
display section 62, a menu section 63, and a status section
64, in like manner as the multitask monitoring screen 61.
When using the template building tool, described herein,
the operator may view the template building screen 71 and
manipulate the commands and elements thereon by means of
a pointing device, such as a mouse. A detailed description of
how the operator may use the template builder tool is given
65
may be skipped entirely.
The maximum time section 96 for a line 92 may specify
a maximum time which the designated process step may
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take, which might be infinity. If the maximum time is
infinity, the system may delay completion of the designated
process step until after all other process steps with finite
maximum time have been completed.
Each line 92 may also have an additional data section 97
for the designated process step, which may specify whether
(1) the step is to be done, (2) the step is to be skipped, or (3)
the process is to be “held” or temporarily halted at the
designated process step for input from the operator. In the
latter case, for example, the process might be “held” at the
designated process step until an operator confirms that the
process should continue.
10
MULTITASKING AND OPTIMIZATION
Having delineated all the steps of all the procedures, the
computer may determine the most efficient manner for
carrying out the procedure. The task would be simple if the
steps of the first process were to be completed before the
apparatus started on the second process. Through the use of
time interleaving, multiplexing or multitasking the computer
is utilized to keep track of multiple operations so as to
perform a number of different processes each having a
multiplicity of steps simultaneously.
In multitasking, a number of samples, each undergoing
separate exposures may all be worked on simultaneously. In
time interleaving, the robotic arm may operate through a
sequence which is determined by the timing of the indi
vidual steps of many processes and the robotic arm trans
ports different samples in a time efficient sequence rather
than a process ordered sequence. Although the robotic
device can only move one sample to a work station at a time,
the entire system is continuously monitoring. Scheduling and
processing all tasks and their times at each station concur
rently. At each step the process performed at that worksta
tion continues (e.g., chemical reactions) even when the
robotic arm is not currently attending to it. In other words,
15
20
25
30
occurs, the robotic device will be scheduled to move to the
35
the sample is disposed in the workstation and the robotic arm
continues to grasp another sample. The process step contin
ues to work on the first sample while the robotic arm is
attending or transporting the second sample. The multiple
process steps that are being done, one to each sample, are
being done in parallel and are not serial processes.
In fact the robotic arm works on a sample for a short
period of time during which it usually transports a sample to
a work station and then leaves that sample and works on
45
processed at a work station.
55
The method carried out by the control station 14 for
multitasking may be described by pseudocode shown in
Tables 4–8 herein. It would be clear to one of ordinary skill
in the art, after perusal of the specification, drawings and
claims herein, that modification of known processor systems
to perform the functions disclosed in this pseudocode (as
well as in other pseudocode disclosed herein) would be a
straightforward task and would not require undue experi
mentation.
TABLE 4
the robotic movements necessary to complete the entire run
of all the steps in all the processes to be run. This determi
nation may be completed before any movement is initiated.
If at any time during the running of the multitasking any
steps are added to one or more of the processes or any of the
steps are reconfigured during the run, a new determination
may be completed wherein the computer recalculates all the
movements necessary to complete the run and insures that
there is no time interference created by the modification to
next step in accordance with the predetermined schedule.
However, if more than one sample is scheduled to move time
arbitration ensues. Time arbitration determines the fuzzy
time window for each of the time conflicting steps and
selects the sample in the most time critical step to move. If
more than one step has a critical time, the computer com
pares the times during the previous movement and varies the
timing of the previous tasks to resolve or prevent bottlenecks
from occurring. In a similar manner a single resource can be
scheduled to work on two different samples during the same
time period and such conflicts can be resolved in a similar
manner using the arbitration method.
PSEUDOCODE FOR MULTITASKING
another sample or samples before returning again to the first
sample. Thus the robotic device work on each sample is
suspended during the time interval that it is working on
another sample or during which the samples are being
The multitasking of the different processes is dependent
upon the instructions issued to the robotic device, relative to
the timing of each of the steps in the multiple processes and
the optimization of the multitasking operations, to move the
samples at the scheduled times determined by the computer
inputs.
The computer control (software) may first determine all
28
the run. This method of predetermining the movements can
of course be replaced by a real time method of determining
movement but it is believed that the predetermining method
is more advantageous. The predetermining method identifies
time conflicts, if any, where the robotic device would be
required to perform two tasks simultaneously, resolves any
such conflicts that may exist, and optimizes the schedule for
the minimum time required to complete the entire run of the
multiple processes.
This method of predetermination employs certain deci
sion making procedures which are designed to permit the
computer to resolve time conflicts and iteratively optimize
the schedule. An iterative optimization method is used
because the complexity of scheduling different multiple
tasks, each with the possibility of having multiple critically
timed steps, is too complex to be solved by using math
ematical techniques. In addition, the decision making rules
allow the resolution of other conflicting requirements for
other resources such as the peripheral equipment or work
station modules, which may be used in conjunction with the
robotic equipment.
As described above, a predetermined schedule may be
developed to resolve time and resource conflicts and the
schedule may be iteratively optimized to minimize the time
required to complete the steps of the multiple processes. In
order to interleave the steps of the multiple processes each
step of each task is examined at predetermined intervals.
e.g., one minute. A calculation is made of the time to
completion of the current step. If the step incubation time is
finished a move condition results. If that is the only move
condition during this time, i.e., only one move condition
Multitasking Data Structure
STRUCTURE TASKARRAY [1500 elements )
BYTE
PROCESS NUMBER;
BYTE
TASK NUMBER;
CHAR [25] TASK NAME;
65
INTEGER
INTEGER
TASK X COORDINATE OF WORKSTATION,
TASKY COORDINATE OF WORKSTATION;
LONG INTEGER ENCODED REAL TIME FOR PICKUP OR
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TABLE 4-continued
TABLE 4-continued
Multitasking Data Structure
5
Multitasking Data Structure
DROPOFF;
CHAR [1]
DROPOFF/PICKUP FLAG;
CHAR [5]
MOVE_FLAG;
CHAR [5]
RESOURCE_FLAG;
{ If set TRUE, two or more tasks require the same
resource. Resource arbitration is done to resolve all
{When TRUE the process flagged needs to move to next
task in progress. This information is entered into the
task array. If multiple flags are set simultaneously the
process steps must be arbitrated. }
10
conflicts. }
TABLE 5
Multitasking (Build Schedule)
PROCEDURE BUILD MULTITASK SCHEDULE ()
{ This routine is called a number of times with different
seeding to build a statistical sampling of a number of
schedules. The calling routine picks the most optimal schedule
to run. }
BEGIN
{ Initialize timer and pick a process for first move. For
iterative tasks, processes will be started in various orders to
seed task builder and establish different scheduling. At each
timer tick all processes are examined to check whether it is
time to move to next position. If TRUE the task will be
entered into the task array at the scheduled time. If more
than one process needs movement at the same timer tick, time
arbitration ensues. If two or more processes need the same
resource, resource arbitration is undergone. This process
continues until all tasks in all processes are complete. }
TIMER = 0;
START FIRST PROCESS;
WHILE NOT ALL PROCESSES STARTED DO BEGIN
INCREMENT TIMER BY 1;
IF ANY TASK NEEDS MOVEMENT THEN
SET TASK MOVE FLAG
ELSE
START NEXT PROCESS;
IF MOVE_FLAG > 1 THEN TIME_ARBITRATE for multiple moves }
IF TASK MOVE THEN ADD TASK TO TASK ARRAY ITASK COUNTER)
END;
WHILE NOT ALL PROCESSES COMPLETED DO BEGIN
INCREMENT TIMER BY 1;
IF ANY PROCESS NEEDS MOVEMENT THEN SET TASK MOVE FLAG;
IF MOVE FLAG > 1 THEN TIME_ARBITRATE for multiple moves }
IF TASK MOVE THEN ADD TASK ARRAY (TASK] for resource use }
END;
END;
TABLE 6
Multitasking (Time Arbitrate)
PROCEDURE TIME_ARBITRATE ()
{If two or more processes must be moved simultaneously, the
times are arbitrated, first by examining fuzzy time range and
adjusting those process tasks with fuzzy time. If the
colliding processes are critically timed the processes’ prior
tasks are rearranged to circumvent the collision. This
procedure is called in REARRANGE_ARRAY (). }
INTEGER
FUZZY_TIME (COMP the comparMAXETIMEa maximum value }
BYTE CRITICAL_FLAG
= 0; £ initialize critical flag #
BYTE CRITICAL FLAG ARRAY [5] = { 0, 0, 0, 0, 0 };
BEGIN
FOR I = 1 TO MAX PROCESSES
IF (PROCESSS [I].MOVE_FLAG_SET AND FUZZY_TIME [I] :
FUZZY_TIME COMP)
THEN BEGIN
TASK MOVE = I; H finds shortest fuzzy time ;
FUZZY_TIME COMP = FUZZY TIME [I];
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TABLE 6-continued
Multitasking (Time Arbitrate)
IF (FUZZY TIME = 0) THEN BEGIN
SET CRITICAL_FLAG;
SET CRITICAL_ARRAY (TASK];
END;
END;
{ If two or more processes need to move immediately a
rearrangement of earlier interleaved tasks occurs to
settle conflicts at this point if a fuzzy time range
settle the conflict the process with the shortest fuzzy
time value is set to move. }
IF CRITICAL_FLAG > 1 THEN REARRANGE_ARRAY ();
ELSE
ADD TASK_ARRAY (TASK MOVE};
END;
TABLE 7
Multitasking (Resource Arbitrate)
PROCEDURE RESOURCE ARBITRATE ()
{ If two or more processes need the same resource (physical
location), fuzzy times for the processes in question are
examined to evaluate whether the time slack can settle the
conflict. If not, the processes prior tasks are rearranged to
circumvent the collision.
BYTE CRITICAL_FLAG
= 0; H initialize critical flag +
BYTE CRITICAL FLAG_ARRAY_[5] = { 0, 0, 0, 0, 0 };
BEGIN
{ Compare process task fuzzy time with other process actual
task time. }
COMPARE CRITICAL_PROCESS_1_FUZZY_TIME WITH
CRITICAL PROCESS_2_TASK TIME;
IF -TASK_MOVE = PROCESS_2;
ELSE
COMPARE CRITICAL_PROCESS_2_FUZZY_TIME WITH
CRITICAL_PROCESS_1_TASK TIME;
IF -TASK MOVE = PROCESS_1;
IF TASK_MOVE TRUE
ADD TASK ARRAY (TASK_MOVE};
ELSE BEGIN
SET CRITICAL_FLAG;
SET CRITICAL_FLAG_ARRAY (TASK];
REARRANGE TASK_ARRAY ();
END;
END;
TABLE 8
Multitasking (Rearrange Tasks)
PROCEDURE REARRANGE TASK_ARRAY ()
{To prevent conflicts which camot be arbitrated with fuzzy
timing the processes in conflict are examined at their previous
step(s) and timing adjusted in that task to remedy the conflict
at the current task. After time adjustment of the critical
process the task array is reset to the newly adjusted position
and returns to the multitask builder and reworks the rest of
the tasks in all processes. }
BEGIN
{Find the last time the critical process was moved. }
REPEAT
POSITION = POSITION – 1;
UNTILTASK ARRAY [POSITION] = CRITICAL_FLAG_ARRAY (TASK];
{Adjust timer. }
INCREMENT TASK ITASK ARRAY [POSITION].MIN_TIME) BY X;
{ Reset position and time. }
SET POSITION TO CURRENT TASK_ARRAY VALUE;
SET TIMER TO CURRENT TASK_ARRAY VALUE;
RETURN TO MULTITASK BUILDER;
END;
5,696,887
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IP an immunoserum, e.g., for enzyme detection
LO WASH a low stringency (low ionic concentration)
wash, typically used for DNA probes
ME BL methylene blue stain
PADxxx a blotter, preferably 9% inch thick
PARK a location to wait until a next step
PROBE a DNA probe
SCHIF Schiff reagent for a Schiff reaction
STN a stain
XY xylene
Those skilled in the art will recognize, after perusal of this
application, that other and further protocol operations,
reagents, chemoactive or bioactive compounds, buffers, or
other substances would be workable with the devices and
substances disclosed herein, and are within the scope and
spirit of the invention.
Alternative Embodiments
36
While preferred embodiments are disclosed herein, many
variations are possible which remain within the concept,
scope, and spirit of the invention, and these variations would
become clear to those skilled in the art after perusal of this
10
application.
For example, it would become clear to those skilled in the
art that the devices and techniques described herein would
be applicable to other processes, subject to standardization
and robotic operation, and that such application would be
within the concept, scope, and spirit of the invention. Such
processes could include those related to developing film and
those related to manufacture or testing of electronic circuits,
printed circuit boards, or semiconductor wafers.
For a second example, it would become clear to those
15
skilled in the art that the devices and techniques described
herein for use with liquid would generally be applicable to
processes using other flowable substances, including
colloids, gels, or powders, and that such application would
be within the concept, scope, and spirit of the invention.
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BioTek Solutions, I
Page 1
Reviewed
Reviewed
Under Revision
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PAS - Periodic Acid Schiff
Histochemical Stain
TechMate
Use protocol PAS which requires a total time of 1 hour, 26 minutes and 25 seconds and has a total of
46 steps.
Principle
Schiff-base reactions were first described by H. Schiff in 1865. These reagents specifically react with
aldehydes to form a colored (stained) product. Periodic acid oxidizes carbon-carbon bonds where the
adjacent carbons are:
1,2-dihydroxy
1-hydroxy-2-keto
1-hydroxy-2-primary amine
1-hydroxy-2-secondary amine
The oxidation of these bonds by periodic acid results in the formation of aldehydes, and these are
demonstrated with Schiff reagent. In cells and tissues, the PAS procedure identifies complex carbo
hydrates, including glycogen.
Specimen
The Periodic Acid Schiff procedure is a histochemical stain, and is both specific and precise. With
adequate control specimens, the resulting stain may be quantitated using an image analysis system.
However, it should be noted that some cellular constituents, notably glycogen, are to a greater or
lesser extent lost during routine tissue processing. The PAS stain is quantitative only ?or the
material present after processing.
(1) The specimen will consist of tissue section, fixed smears, or fixed, cultured cells adhered to glass
slides.
(2) 10% neutral buffered formalin is the preferred fixative, although others may be used. Calcium
formol has been recommended to assist in the preservation of glycogen. PAS can be used following
most routine fixatives, including Bouin's, Carnoy's and Zenker's.
f/-.... . . 2/
fºr * , .!. +
ix. + - C -
A-1 #e
. . . . , 1, 5
ºr , 4, 25, **
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Bietek Solations, {ng
Page 2
(3) Counterstaining is generally performed using hematoxylin, since most PAS - positive structures
are cytoplasmic.
Specimen Preparation
A. All specimens must be mounted away from the label end of the slide. It is important that
specimens be placed on the side of the slide that hols the painted babel area, that is, the "painted" area
should be up. If tissues mist be stained on the TechMate that have not been mounted on Capillary
Gap Pius slides, a yellow BioTek 130 micron slide (blank) may be used to face the non-capillary Gap
slide to create a the gap.
B. Paraffin sections should be mounted from a preheated water bath containing distilled or
deionized water. The water bath should contain no additives (such as gelatin, polylysine, etc.).
Sections should be dried by heating, generally at approximately 60 C for a minimum of 60 minutes.
It is important to drain water from beneath the section prior to the oven drying process.
C. Slides containing smears must contain no large clumps or other accumulations of material
that cause “humpiness” of the smear surface. Any clumps of material will be toothick for adequate
microscopic visualization, and will interfere with capillary gap staining.
D. Smears or cultured cell preparations should be fixed prior to staining. If the fixative
leaves a residue on the slides, they may require alcohol steps prior to staining. Fixed, air-dried smears
or cultures that do not have a fixative residue (such as carbowax or polyethylene glycol) may be
stained starting with the first aqueous buffer steps of the protocol. It is preferable to prewet the slides
in buffer in a Coplinjar prior to loading into the TechMate shile holder. This wetting should be done
with TechMate buffer 2 or 3, for several minutes. This pre-wetting will ensure adequate fluid flow in
the capillary gap when staining is started in a truncated protocol at an aqueous step.
and may demonstrate a fixation"flow” artifact, in which staining is heavier on one side of each cell
than the other. This flow artifact indicates the penetration pathway of fixatives and processing sol
vents.
Preparation of Reagents
Periodic Acid,0.5%: 0.5gm periodic acid in 100 ml deionized H.O.
Schiff Reagent: Commercial preparation such as Accra Lab (cat. #25043).
Schiff reagent may be prepared in the laboratory using the methods described in the AFIP
Bleaching Solution: 0.5% sodium metabisulfite (Na,S.O.). Make up fresh daily.
Hematoxylin: BioTek progressive-staining hematoxylin.
Procedure
Use protocol PAS on TechMate.
Critical steps in this procedure are the preparation of the periodic acid, and the time of acid exposure.
The source and type of Schiff reagent is non-critical, as is the length of exposure to the Schiff reagent.
While the general standard is use of a basic Schiff reagent (resulting in Magenta-colored positive
staining) any Schiff reagent of any particular color may be used. If any other color Schiff reagent
than the standard magenta is used, this fact must be brought to the attention of the individual who will
evaluate the slides.
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BioTek Solutions, lºg
PAS, continued
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Bigfek Solutions, Inc = Page
Mop Templo?e for Protocol
PAS
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IPABC Protocol
Standard Immunoperoxidase Staining with
Avidin-Biotin-Complex Detection System
TechMate
Use Protocol IPABC which requires a total time of 4 hours, 15 minutes and 38 seconds and
has a total of 94 steps.
Principle
The IPABC protocol utilizes a type of immunostaining referred to as a “sandwich" technique,
in which a specimen is first reacted with an unlabelled primary antibody. Due to the specificity
of the antibody for the desired antigens (epitopes), the primary antibody will react with these
antigens, forming an antigen-antibody complex. In the second functional step of the protocol,
a secondary antibody directed against the species of the primary antibody is applied to the
specimen. This secondary antibody is biotinylated, that is, the antibody contains one or more
biotin molecules that have been chemically attached to the secondary antibody during its
manufacture. The secondary antibody recognizes any primary antibody that has bound to the
specimen. In the third functional step of the protocol, a complex of avidin and biotin, which is
also complexed with peroxidase enzyme, is permitted to react with the specimen. This com
plex binds to any sites that have previously reacted with the primary and secondary antibody.
The fourth functional step of the protocol then provides a chromogenic substrate for the
peroxidase enzymes of the avidin-biotin-complex. The result of enzyme activity on the chro
mogenic substrate is to render the soluble chromogen into a colored, insoluble product which
precipitates at sites of enzyme activity. This series of functional steps results in deposition of
color at microscopic sites of specimens that contain the specific epitopes recognized by the
primary antibody.
Specimen
(1) The specimen will consist of tissue sections (paraffin), fixed smears, or cultured cells
adhered to glass slides.
(2) A positive control slide will be run each and every time the stain is performed.
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tions, Inc
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3) A variety of fixatives may be used for specimen preservation. However, it should be under
stood that all fixation procedures result in some reduction of antigen recognition. Formalin
fixation should be kept to a minimum, and the laboratory should test each primary antibody
used in a fixation test protocol to verify that the fixation of the specimen is not creating nega
tive specimens from positive ones.
Specimen Preparation
A. All specimens should be mounted on Probe-On Plus glass slides. Specimens must be
mounted away from the label end of the slide. It is important that specimens are placed on the
side of the slide that holds the painted label area, that is, the “painted” area should be up. If
tissues must be stained on the TechMate that have not been mounted on Probe-On Plus slides,
a yellow 130 micron slide (blank) may be used to face the non Probe-On Plus slide to create a
capillary gap.
B. Paraffin sections should be mounted from a preheated water bath containing distilled
or deionized water. The water bath should contain no additives such as gelatin, polylysine,
etc. Sections should be dried by heating, generally at approximately 60°C for a minimum of 60
minutes. It is important to drain water from beneath the section prior to the oven drying
process.
C. Slides containing smears must contain no large clumps or other accumulations of
material that cause “lumpiness” of the smear surface. Any clumps of material will be too thick
for adequate microscopic visualization, and will interfere with capillary gap staining.
D. Smears or cultured cell preparations should be fixed prior to staining. If the fixative
leaves a residue on the slides, they may require alcohol steps prior to staining. Fixed, air-dried
smears or cultures that do not have a fixative residue (such as carbowax or polyethylene
glycol) may be stained starting with the first aqueous buffer steps of the protocol. It is prefer
able to prewet the slides in buffer in a Coplin jar prior to loading into the TechMate slide
holder. This wetting should be done with TechMate buffer 2 or 3, for several minutes. This
pre-wetting sill insure adequate fluid flow in the capillary gap when staining is started in a
truncated protocol at an aqueous step.
Preparation of Reagents
100% Absolute Alcohol
100% Ethanol................
-------
----------------------------------------- 20 mls near End
This reagent is commercially purchased and stored at room temperature in an approved
safety cabinet.
Xylene(s)
Xylene........................................…..... 20 mls at start
--------------------------------------------------20 mls near end
Note: Xylene at end should be in HOME position
This reagent is commercially purchased and stored at room temperature in an approved
safety cabinet.
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Page 3
Water Wash
Water Wash .............................................25 mls
This reagent is commercially purchased from BioTek Solutions, Inc.
Storage directions
are listed on the container.
Buffer Solution Number 1,2 and 3 (BUF 1-3)
Buffer Solutions........................................25 mis
These solutions are commercially purchased from BioTek Solutions, Inc. Storage directions
are listed on the container.
BioTek Iodine
BioTek Iodine.......................................... 10 mls
This reagent is commercially purchased from BioTek Solutions, Inc.
Storage directions
are listed on the container.
BioTek Hypo (thiosulfate)
BioTek HYPO ......................................... 10 mls
This reagent is commercially purchased from BioTek Solutions, Inc.
Storage directions
are listed on the container.
Hydrogen Peroxide (HP)
HP Block.................................................. 10 mls
Hydrogen Peroxide Block solution is commercially purchased from BioTek Solutions, Inc.
Storage conditions are listed on the container.
Blocking Antibodies (BLOK)
Blocking Antibody......................................8 drops per individual slide well.
This serum is commercially purchased from BioTek Solutions, Inc.
Primary Antibodies (AB1)
antibodies (prediluted) re purchased from BioTek Solutions, Inc., or are purchased
from other sources. Storage and expiration dates are listed on the container.
Secondary Antibodies (AB2)
Secondary antibodies................................. 8 drops per individual slide well.
Secondary antibody is purchased from BioTek Solutions, Inc. Storage and expiration
dates are listed on the container.
Avidin-Biotin-Enzyme Complex Reagent (ABC)
ABC Reagent...............
.................. 10 drops per individual slide pair well. ABC is
purchaejºonsistersonnion, in
storage inderinondnesareised on the
container(s). (Preparation instructions are given below)
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Page 4
ABC Reagent Preparation
Volume of ABC Required
Chemistry
25 ml
ABC Reagent "A"
50 ml
60 mi
20 drops
Mix thoroughly and let incubate for at least 30 minutes before aliquoting.
Date container and use within 5 days.
DAB Reagent (DAB - Three, 3'diaminobenzidine tetrahydrochloride)
DAB Reagent .................................................750 microliters per individual slidepair
well. DAB is purchased from BioTek Solutions, Inc. Storage and expiration dates are
listed on the container.
DAB Preparation
Volume of DAB Desired
5 ml
25 ml
50 m
60 ml
5 mi
25 ml
50 ml
60 ml
500 ul
1000 ul
1200 ul
500 ul
1000 ul
1200 ul
Mix Thoroughly before Aliquoting.
Mark the Date on the Container and Use Within 5 Days
BioTek Hematoxylin (STN)
BioTek Hematoxylin....................... 10 mls
This stain is commercially purchased from BioTek Solutions, Inc. Storage conditions are
listed on the container.
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Page 5
Procedure
Use protocol IPABC on TechMate.
Load reagents into disposable dishes on TechMate according to reagent template and antibody
positioning sheet. Take care to load proper reagents and volumes for each reagent. Use particular
caution when loading primary reagents. It is critical that each slide pair receive each reagent step.
Refer to general instructions for additional information on organizing your immunoperoxidase stain
ing ?un.
Quality Assurance
All staining runs should include known positive comtrol specimens to validate reagent integrity and
proper performance of the sequential staining steps. When stain results are qualitatively or
quantitatively discordant with expected findings, based on hematoxylin and eosin slides, cytologic
findings, or other clinical data, further evaluation will be performed to resolve any discrepancy at the
discretion of the pathologist, and this will be documented.
Results
Sites within the specimen which contain the particular epitopes (antigenic sites) recognized by the
primary antibody will appear brown. When the stain is performed under controlled conditions, as on
the TechMate, the density of brown color is directly related to the amount of antigen present, Nuclei
will have a hematoxylin counterstain. This hematoxylin will have a purple color, since it is not blued
in this protocol. If a “blued" hematoxylin is desired, substitute buffer 3 in the H2O steps in the
protocol.
References
Brigati, D., L.R. Budgeon, E.R. Unger, et al., Immunocytochemistry is Automated: Development of
a Robotic Workstation based Upon the Capillary Action Principle. J. Histotech. 11: 165-183,
1988.
DeLellis, R.A., et al., immunoperoxidase Techniques in Diagnostic Pathology; Report of a Workshop
by the National Cancer
Institute. Amer. J. Clin. Path, 71: 483- , 1979.
Guesdon, J., et al., The use of Avidin-Biotin interaction in immunoenzymatic techniques. J.
Histochem. Cytochem. 27: 1131- , 1979.
Taylor, C.R., Immunomicroscopy: A diagnostic tool for the surgical pathologist. W. B. Saunders,
Philadelphia, PA 1986.
Larsson, L-I., Tissue Preparation Methods for Light Microscopic Immunohistochemistry. App.
Immunohistochem. 1:2-16, 1993.
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IPABC PROTOCOL
Step Name
XY
PAD1
Min Time
00:05:00
00:00:20
Max Time
00:10:00
00:00:20
Y/NAH
Yes
Yes
XY
PAD1
00:05:00
00:00:20
{}0:10:00
00:00:20
XY
PAD1
00:05:00
{}0:10:00
00:00:20
00:05:00
00:00:20
00:05:00
00:00:20
00:00:15
{X}:00:30
O0:00:#5
00:00:30
Q0:01:30
00:00:30
Ö0:01:30
O0:00:30
00:03:00
(X}:00:30
00:00:20
00:10:00
00:00:20
00:10:00
{{}:00:28
00:10:00
00:00:30
00:10:00
OO:0:0:30
00:01:30
00:00:30
00:01:30
00:00:30
00:10:00
00:00:30
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Q0:00:15
UO: KO:00
00:00:29
00:00:15
00:00:45
00:00:29
00:10:00
00:00:45
Ö0:15:00
00:20:00
00:00:29
{}{}:00:30
00:00:29
00:00:15
00:00:29
00:00:15
00:00:29
00:00:15
00:09:45
{}0:15:00
00:00:29
00:00:15
00:00:45
00:45:00
00:00:29
00:00:15
00:00:29
00:00:30
00:00:29
00:00:15
00:00:29
00:00:15
00:00:29
00:00:15
00:00:45
00:30:00
00:00:29
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
{|0:00:45
00:20:00
00:00:29
00:00:15
00:00:45
00:45:00
00:00:29
00:00:15
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:45
00:30:00
00:00:29
XY
PAD1
XY
PAD1
100%
PAD}
100%
PAOl
IO
PAD1
IO
PAD1
HYPO
:
PADI
H2O
PAD2
H2O
PAD2
HP
PAD2
H2O
PAD2
BUFl
PAD2
BUF1
PAD?
BUF1
PAD2
BLOK
PAD2
BUF1
PAD2
AB1
PAD2
BUF1
PAD2
BUF1
PAD3
BUFl
PAD3
BUFI
PAD3
BUF1
PAD3
A82
PAD3
Yes
Yes
Yes
"Yes
Yes
Yes
Yes
Yes
Yes
Ye?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
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Bie?ek Solutions. Ins
IPABC (continued)
53
54
55
56
53
54
55
56
57
BUF2
PAD3
BUF2
PAD3
BUF2
PAD3
BUF2
PAD3
BUF2
55
PAD3
59
60
6]
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
81
§2.
83
84
BUF2
PAD3
BUF2
PAD 4
ABC
PAD3
sufz
PAD4
BUF3
PAD4
BUF3
PAD4
BUF3
PAD4
BUF3
PAD 4
DAB
PAD4
BUF3
BUF3
Pads
DAB
PAD4
85
86
87
$8
89
H2O
PAD5
H2O
PAD 5
STN
90
91
92
PAD4
H2O
PAD5
93
H2O
94
95
96
PAD 5
100%
PAD5
97
93
99
100
100%
PAD5
100%
PAD5
HOME
00:00:15
00:00:29
00:00:30
00:00:29
00:00:15
00:00:29
00:00:30
00:00:29
00:00:15
Q0:00:29
Ö0:00:15
Q0:00:29
{}0:00:15
(X):00:45
00:30:00
O0:00:29
00:00:15
00:00:29
00:00:30
00:00:29
00:00:15
00:00:29
00:10:00
00:00:29
00:00:15
00:00:15
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:45
00:07:00
00:00:29
00:10:00
00:10:00
00:00:29
00:00:15
O0:00:29
00:00:15
00:00:45
00:07:00
00:00:29
00:00:30
00:00:30
00:00:45
00:07:00
00:00:29
00:00:30
00:00:29
00:00:15
00:00:29
00:04:30
00:00:29
00:02:00
00:00:29
00:02:00
00:00:29
00:00:15
00:00:29
00:00:15
00:00:29
00:00:15
00:00:29
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:45
00:30:00
00:00:29
00:00:15
00:00:29
00:10:00
00:00:29
00:00:45
00:07:00
00:00:29
60:10:00
00:00:29
00:10:00
00:00:29
00:04:30
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
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Page 8
C-2
MAP TEMPLATE FOR PROTOCOL
IPABC
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C-:
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t-l
CONFIDENTIAL:
Adopted
—
resuminºumentation Hºi-L
RELEASE
eviewe
-
Reviewed
Reviewed T
|Under Revision
MAP and 2MAP Protocols
1. Protocol MAP: For 60 Place Slide Holder
2. Protocol 2MAP: For 20 Place Slide Holder
Microwave Antigen Retrieval and Standard
Immuno-Alkaline Phosphatase Staining Using Alkaline
Phosphatase in an Avidin-Biotin-Complex
Detection System
TechMate
1. For 60 Place Slide Holder. Follow all instructions given below through Results section on Page 6.
Use Template MAP on Page 9 with Protocol MAP and 2MAP (Pages7-8) which requires a total
time of 2 hours, 29 minutes and 27 seconds and has a total of 76 steps.
2. For 20 Place Slide Holder. Follow all instructions given below through Results section on Page 6.
Use Template 2MAP on Page 10 with Protocol MAP and 2MAP (Pages 7-8) requiring a total time
of 2 hours, 29 minutes and 27 seconds and has a total of 76 steps.
(Note: Protocols are identical-only the templates differ)
Principle
The MAP and 2MAP protocol utilizes a type of immunostaining referred to as a “sandwich" tech
mique, by which a specimen is first reacted with an unlabelled primary antibody. Due to the specific
ity of the antibody for the desired antigens (epitopes), the primary antibody will react with these
antigens, forming an antigen-antibody complex. In the second finctional step of the protocol, a
secondary antibody directed against the species of the primary antibody is applied to the specimen
This secondary antibody is biotinylated, that is, the antibody contains one or more biotin molecules
that have been chemically attached to the secondary antibody during its manufacture. The secondary
antibody recognizes any primary antibody that has bound to the specimen. In the third functional step
of the protocol, a complex of avidin and biotin that is also complexed with alkaline phosphase enzyme
(alkaline phophatase) is permitted to react with the specimen. This complex binds to any sites that
have previously reacted with the primary and secondary antibodies. The fourth functional step of the
protocol then provides a chromogenic substrate for the alkaline phosphatases of the avidin-biotin
complex. The result of alkaline phosphatase activity on the chromogenic substrate is to render the
soluble chromogen into a colored, insoluble product that precipitates at sites of alkaline phosphatase
activity. This series of functional steps results in deposition of color at microscopic sites of specimens
that contain the specific epitopes recognized by the primary antibody.
This protocol incorporates a microwave pre-treatment of the slides in citrate buffer. This procedure
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restores the immunoreactivity of many epitopes that have been masked or distorted by tissue fixation
protocols.
Specimen
(1) The specimen will consist of tissue sections (paraffin), fixed smears, or cultured cells adhered to
glass slides (a different protocol is provided for cytospins. CAP)
(2) A positive control siide will be run each and every time the stain is performed.
(3) A variety of fixatives may be used for specimen preservation. However, it should be understood
that all fixation procedures result in some reduction of antigen recognition. Formalin fixation should
be kept to a minimum, and the laboratory should test each primary antibody used in a fixation test
protocol to verify that the fixation of the specimen is not creating negative specimens from positive
ones.
Specimen Preparation
A. All specimens should be mounted on ChemMate" Capillary Gap Plus glass slides Speci
mens should be mounted away from the label end of the slide. It is important that specimens are
placed on the side of the slide that holds the painted label area, that is, the “painted" area should be
up. If tissues that have not been mounted on such slides must be stained on the TechMate, pair them
with ChemMate" 130 micron slides (blank) to create the capillary gap. ChemMate" 130 slides
have a YELLOW painted label.
B. Paraffin sections should be mounted from a preheated water bath containing distilled or
deionized water only. The water bath must contain no additives (sych as gelatin, polylysine, etc.).
Sections should be dried by heating, generally at approximately 60 Cfor a minimum of 60 minutes.
It is important to drain water from beneath the section prior to the oven drying process.
C. Slides containing smears must contain no large clumps or other accumulations of material
that cause “lumpiness” of the smear surface. Any clumps of material will be toothick for adequate
microscopic visualization, and will interfere with capillary gap staining.
D. Smears or cultured cell preparations should be fixed prior to staining. If the fixative
leaves a residue on the slides, they may require alcohol steps prior to staining. Fixed, air-dried smears
or cultures that do not have a fixative residue (such as carbowax or polyethylene glycol) may be
stained starting with the first aqueous buffer steps of the protocol. It is preferable to prewet the slides
in buffer in a Coplinjar prior to loading into the TechMate slide holder. This wetting should be done
with ChemMate Buffer 2 or 3, for several minutes. Pre-wetting will ensure adequate fluid flow in
the capillary gap when staining is started in a truncated protocol at an aqueous step.
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— Page 3
rºl
sº
Preparation of Reagents
Microwaving Buffer
BioTek 10X Microwaving Buffer.....................................20 mls
Distilled or deionized water............................................ 180 mls
The 10X microwave buffer stock is purchased from BioTek Solutions, Inc.
For specific instructions on use of the microwave procedure, refer to the general instructins
on microwaving.
100% Absolute Alcohol
100% Ethanol..................................................................25 mls per 10 well tray
This reagent is commercially purchased and stored at room temperature in an approved safety
cabinet.
Buffer solutions Numbers 1,2,and 3 (BUF 1,2,3}
Buffer Solutions................................................................25 mls per 10 well tray
These solutions are commercially purchased from BioTek Solutions, Inc.
Storage directions and expiration date are listed on the containers.
Blocking Antibodies (BLOK)
Blocking Antibody..........................................................8 drops per individual well
This serum is commercially purchased from BioTek Solutions, Inc.
Storage directions and expiration date are listed on the container.
Primary Antibody (AB1)
Primary Antibody..........................................................8 drops per individual well
Primary antibodies (predilute) are purchased from BioTek Solutions, Inc. or from other
sources.
Storage directions and expiration date are listed on the container.
Secondary Antibodies (AB2)
Secondary Antibodies...................................................8 drops per individual well
Secondary antibody is purchased from BioTek Solutions, Inc.
Storage directions and expiration date are listed on the container.
Avidin-Biotin-Alkaline Phosphatase Complex Reagent (AP)
AP Reagent”..................................................................325 pil (or 8 drops) per individual well
AP is purchased from BioTek Solutions, Inc.
Storage directions and expiration date are listed on the container.
* (AP preparation directions continued overpage)
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AP Reagent Preparation
Volume of AP Desired
Phosphate Buffered Saline (PBS)
5 ml ºn son
AP-ABC Reagent “A.”
tº sºlº
AP-ABC Reagent “B”
1 drop
on
5 drops 10 drops | 12 drops
Mix thoroughly and let incubate for at least 30 minutes before aliquoting.
Date container and store at 4 degrees C. Use within 5 days.
BioTek Hematoxylin (STN)
BioTek Hematoxylin............................................ 10 mls per 10 well tray
This stain is commercially purchased from BioTek Solutions, Inc.
Storage conditions and expiration date are listed on the container.
BT Red Reagent (a New Fuchsin-type chromogen)
Prepare just before use? Approximately 15 minutes before the
slides reach the chromogen position.
BT Red Reagent.................................................750 pul per individual slide well.
BT Red is purchased from BioTek Solutions, Inc. Storage and expiration dates are listed on
the container(s). This chromogen is fluorescent, as well as producing an absorbing (visible)
product. This chromogen may also be dehydrated prior to coverslipping. If aqueous media
are used for coverslipping, rehydrate the slides from the final 100% alcohol step prior to
using the aqueous mounting medium. Failure to include the alcohol step may result in the
appearance of crystals over the chromogen deposits.
If the specimen contains endogenous alkaline phosphatase, it may be necessary to block this
prior to chromogen development. This may be done by including Levamisole in the BT Red
chromogen (Levamisole is included as a component in the BT Red chromogen kit). Use
Levamisole only if required. For those specimens that do require blocking of endogenous
compound BT Red reagent. Add and mix before adding BT Red components. After
alkaline phosphatase, add one drop of Levamisole to each 10 ml of Tris Buffer used to
mixing, discard any volume that will not be used for making BT Red reagent.
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Page 5
Chemistry
Volume of BT Red Required
Mix thoroughly before aliquoting.
Use within 15 minutes.
Using the value of 750 ul of reagent per slide pair, I slide pair will require 750 ul of reagent; 2 slide
pairs will require 1.5 ml, 10 slide pairs will require 7.5 ml; 20 slide pairs will require 15 ml. A full
slide carrier of 30 slide pairs will require 22.5 ml.
Procedure
Use protocol MAP and 2MAP on TechMate.
Prior to starting TechMate, slides must be organized for the staining run, deparaffinized, hydrated to
buffer (water), and subjected to microwaving in BioTek Microwaving Buffer. Detailed instructions
for these steps are given in the general microwaving procedures. It is critically important to follow
the microwaving procedures precisely, in order to insure reproducible retrieval of antigenic sites. At
the conclusion of the microwaving step, slides must be cooled to room temperature. During this cool
down interval, the TechMate can be prepared for the staining run.
Load reagents into disposable dishes on TechMate according to reagent template and antibody posi
tioning sheet. Take care to load proper reagents and volumes for each reagent. Use particular
caution when loading primary reagents. It is critical that each slide pair receive each reagent step.
Refer to general instructions for additional information on organizing your immunoperoxidase stain
ing run.
Quality Assurance
All staining runs should include known positive control specimens to validate reagent integrity and
proper performance of the sequential staining steps. When stain results are qualitatively or quantita
tively discordant with expected findings, based on hematoxylin and eosin slides, cytologic findings, or
other clinical data, furtherevaluation will be performed to resolve any discrepancy at the discretion of
the Pathologist, and this will be documented.
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p. 3
i.e.,
Page 6
Results
Epitopes (antigenic sites) recognized by the primary antibody will appear RED. The density of red
color is directly related to the amount of antigen present, when the stain is performed under con
trolled conditions, as on the TechMate. Nuclei will have a hematoxylin blue counterstain.
References
Brigati, D., L.R. Budgeon, E.R. Unger, et al., Immunocytochemistry is Automated: Development of
a Robotic Workstation based Upon the Capillary Action Principle. J. Histotech. 11: 165-183,
1988.
DeLellis, R.A., et al., Immunoperoxidase Techniques in Diagnostic Pathology; Report of a Work
shop by the National Cancer Institute. Amer. J. Clin. Path. 71: 483-488, 1979.
Guesdon, J., et al., The use of Avidin-Biotin interaction in immunoenzymatic techniques. J.
Histochem. Cytochem. 27: 1131-1139, 1979.
Larsson, L.-I., Tissue Preparation Methods for Light Microscopic Immunohistochemistry. App.
Immunohistochem. 1:2-16, 1993.
Shi, S.R., M.E. Key and K.L. Kalra, Antigen Retrieval in Formalin-fixed Paraffin-embedded Tissues:
An Enhancement Method for Immunohistochemical Staining Based on Microwave Oven
Heating of Tissue Sections. J. Histochem. Cytochem. 39:741-748, 1991.
Shi, S.R., C. Cote, K.L. Kalra and C.R. Taylor. A Technique for Retrieving Antigens in Formalin
fixed, Routinely Acid-decalcified, Celloidin-embedded Human Temporal Bone Sections for
Immunohistochemistry. J. Histochem. Cytochem. 40: 787-792, 1992.
Suurmeijer, A.J.H. and M.E. Boon. Optimizing Keratin and Vimentin Retrieval in Formalin-fixed,
Paraffin-embedded Tissue with the Use of Heat and Metal Salts. App. Immunohistochem. 1:
143–148, 1993.
Taylor, C.R., Immunomicroscopy: A diagnostic tool for the surgical pathologist. W. B. Saunders,
Philadelphia, PA 1986.
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Cºmº
MAP and 2MAP Protocol
Step
Step Name
Min Time
Max Time
Y/NIH
i
2
3
4
5
§
7
BUF2
PAD
BUFi
PAD)
BUFi
PAD |
BLOK
{}0:00: # 0
00:00:29
00:00:10
00:00:29
Ö0:00: 10
00:00:45
00:07:00
()0:10:00
00:00:29
O0:10:00
00:00:29
00:10:00
00:00:45
00:10:00
Yes
Yes
Yes
Yes
Yes
Yes
Yes
8
9
10
11
|2
I3
14
15
16
17
18
19
20
21
22
PADI
BUF
PADI
AB!
PAD1
O0:00:29
00:00:10
00:00:45
00:25:00
00:00:29
00:00:29
00:00:10
00:00:45
00:25:00
00:00:29
Yes
Yes
Yes
Yes
Yes
BUFI
PADI
BUFI
PAD !
BUF1
PAD?
BUF
PAD2
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:45
00:00:10
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
Yes
Yes
Yes
Ye?
Yes
Yes
Yes
Yes
00:10:00
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:10:00
00:00:29
Ö0:10:00
Yes
Yes
Yes
Yes
Ye?
Yes
Yes
Yes
Yes
00:00:29
00:10:00
Ye?
Yes
00:00:29
00:10:00
00:00:45
00:25:00
O0:00:29
00:00:10
00:00:29
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Ye?
Ye?
23
24
25
26
27
28
29
30
3i
32
33
34
35
36
37
38
39
40
BUF1
PAD2
AB2
PAD2
BUFl
PAD2
BUF2
PAD2
BUF2
PAD2
BUF2
PAD2
BUF2
PAD2
AP
PAD2
BUF2
PAD3
4:
42
43
44
BUF3
PAD3
BUF3
PAD3
BUF3
PAD3
45
46
47
48
49
50
00:25:00
O0:00:29
Q0:00:10
O0:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00; jø
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00: 10
00:00:29
O0:00: 10
00:00:29
00:10:00
00:00:29
00: 10:00
00:00:29
00: 10:00
00:00:29
BUF3
PAD3
CHROM GEN
PAD3
00:00:30
00:00:45
00:07:00
00:00:29
00:10:00
60:00:45
00:07:00
00:00:29
Yes
Yes
Yes
Yes
BUF3
PAD3
00:00: 10
00:00:45
00:05:00
{}{}:00:45
Yes
Ye?
Yes
Yes
Ye?
Ye?
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Template for Protocol
MAP
CHROM
GEN
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tº-3
TEMPLATE FOR PROTOCOL
2MAP
ºur
PAD2
PAD 4
| Ho HE CHRO
alok II snº
* These positions cannot be used
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fºl
Page |
$ºis...,as [Adopted
ople
PRELIMINARY DOCUMENTATION
RELEASE
Reviewed
|Reviewed
|
|
|
|
T
||
Reviewed tº
Reviewed TT
T.
Under Revision||
||
|
MIP-AP and 2MIP-AP PROTOCOLS
1. Protocol MIP-AP for 60 Place Slide Holder
2. Protocol 2MIP-AP for 20 Place Siide Holder
Microwave Antigen Retrieval, and Double Immunostaining
Using Peroxidase and Alkaline Phosphatase in
Avidin-Biotin-Complex Detection Systems
TechMate"
1. For 60 Place Slide Holder: Follow the instructions through the Results section (Page 6). Use
Protocol MIP-AP (Page 7-9) which requires a total time of 4 hours, 42 minutes and 42 seconds and
has a total of 138 steps, and the MIP-AP Template (Page 10).
2. For 20 Place Slide Holder: Follow the instructions through the Results section (Page 6). Use
Protocol 2MIP-AP (Pages 7-9) (time as noted above in #1), and the 2MIP-AP Template (Page 11).
(Note: Protocols are identical - only the templates differ.)
Principle
The MIP-AP and 2MIP protocols utilize a type of immunostaining referred to as a “sandwich" tech
nique, in which the specimen is first reacted with an unlabeled primary antibody. Due to the specific
ity of the antibody for the desired antigens (epitopes), the primary antibody will react with these
antigens, forming an antigen-antibody complex. In the second functional step of the protocol, a
secondary antibody directed against the species of the primary antibody is applied to the specimen.
This secondary antibody is biotinylated, that is, the antibody contains one or more biotin molecules
that have been chemically attached to the secondary antibody during its manufacture. The secondary
antibody recognizes any primary antibody that has bound to the specimen. In the third functional step
of the protocol, a complex of avidin and biotin that is complexed with peroxidase enzyme (peroxi
dase) is permitted to react with the specimen. This complex binds to any sites that contain biotin (the
sites that had previously reacted with the primary and secondary antibody). The fourth functional
step of the protocol then provides a chromogenic substrate for the peroxidase of the avidin-biotin
complex. The result of peroxidase activity on the chromogenic substrate is to render the soluble
chromogen into a colored, insoluble product that precipitates at sites of enzyme activity. The fifth
functional step consists of exposing the specimen to another unlabelled primary antibody that is
directed against a different group of antigens. The biotinylated secondary antibody is then applied
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again in the sixth functional step, thus recognizing the new (second) primary antibody that has bound
to the specimen. In the seventh functional step of the protocol, a mixture of avidin and biotin which
is complexed with alkaline phosphatase enzyme (alkaline phosphatase) is permitted to react with the
specimen. After the avidin-biotin-complex has bound to sites that have previously reacted to the
second application of the primary and secondary antibodies, a chromogenic substrate for alkaline
phosphatase is introduced. This series of functional steps results in the deposition of two contrasting
colors at microscopic sites of specimens that contain the specific epitopes recognized by each primary
antibody. With the substrates used in this protocol, the sites binding primary antibody labelled with
peroxidase will appear brown as a result of the chromogen DAB, and the sites binding primary
antibody labelled with alkaline phosphatase will appear red as a result of the chromogen BioTek Red.
This protocol also utilizes a pre-treatment in a citrate buffer in a microwave oven. This procedure
restores the immunoreactivity of many epitopes which are masked or distorted by tissue fixation
protocols.
Specimen
(1) The specimen will consist of tissue sections (paraffin), fixed smears, or cultured cells adhered to
glass slides.
(2) A positive control slide will be run each and every time the stain is performed.
(3) A variety offixatives may be used for specimen preservation. however, it should be understood
that all fixation procedures result in some reduction of antigen recognition. Formalin fixation should
be kept to a minimum, and the laboratory should test each primary antibody used in a fixation test
protocol to verify that the fixation of the specimen is not creating negative specimens from positive
oncs.
Specimen Preparation
A. All specimens should be mounted on ChemMate" Capillary Gap Plus glass slides. Speci
mens should be mounted away from the label end of the slide. It is important that specimens be
placed on the side of the slide that holds the painted label area: that is, the “painted" area should be
up. If tissues must be stained on the TechMate that have not been mounted on such slides, pair
ChemMate"Capillary Gap 130 micron slides (blank) with them to create the capillary gap. The
ChemMate" 130 micron slides have YELLOW painted labels.
B. Paraffin sections should be mounted from a preheated water bath containing distilled or
deionized water. The water bath should contain no additives such as gelatin, polylysine, etc. Sec
tions should be dried by heating, generally at approximately 60°C for a minimum of 60 minutes. It is
important to drain water from beneath the section prior to the oven drying process.
C. Slides containing smears must contain no large clumps or other accumulations of material
that cause “lumpiness” of the smear surface. Any clumps of material will be toothick for adequate
microscopic visualization, and will interfere with capillary gap staining.
D. Smears or cultured cell preparations should be fixed prior to staining. If the fixative leaves
a residue on the slides, they may require alcohol steps prior to staining. Fixed, air-dried smears or
cultures that do not have a fixative residue (such as carbowax or polyethylene glycol) may be stained
with no pretreatment.
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Preparation of Reagents
Microwaving Buffer
BioTek Microwaving Buffer..........20 mls
Distilled or deionized water......... 180 mls
This microwave buffer stock is purchased from BioTek solutions, Inc. For specific
instructions on use of the microwave procedure, refer to the general instructions on
microwaving.
100% Absolute Alcohol (100%)
100% Ethanol ..........................................25 mls per 10 well tray
This reagent is commercially purchased and stored at room temperature in an approved
safety cabinet.
Buffer Solution Number 1A, 1B, 2A, 2B, 3A, and 3B
Buffer Solutions......................................25 mls per 10 well tray
These solutions are commercially purchased from BioTek Solutions, Inc. Storage conditions
are listed on the container. Note: The “A” and “B” designations refer to the same buffer,
either 1, 2 or 3. An A and a B buffer are used simply to supply the additional quantities of
buffer used in this protocol.
Water Wash (H2O)
BioTek Water Wash Solution..................25 mls per 10 well tray
This reagent is commercially purchased from BioTek Solutions, Inc.
Storage conditions and expiration date are listed on the containers.
HP Block...…. 10 mls per 10 well tray
Hydrogen Peroxide Block solution is commercially purchased from BioTek solutions, Inc.
Storage conditions and expiration dates are listed on the container.
Blocking Antibodies (BLOK)
This serum is purchased from BioTek Solutions, Inc.
Storage conditions and expiration dates are listed on the container.
Avidin-Biotin-Enzyme Complex Reagent (ABC)
ABC Reagent............................................350 ul (or 8 drops) per individual slide well.
ABC is purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container(s).
ABC Reagent Preparation
Chemistry
Volume of ABC Desired
5 ml
Phosphate Buffered Saline (PBS)
ABC Reagent “A.”
20 drops. 40 drops | 48 drops
ABC Reagent “B”
20 drops
48 drops
Mix thoroughly and let incubate for at least 30 minutes before aliquoting.
Date container and store at 4 degrees C. Use within 5 days.
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Primary Antibodies (AB1A and AB 1B)
Primary Antibody...................................8 drops per individual slide well.
Primary antibodies (predilute) are purchased from BioTek Solutions, Inc., or are purchased
from other sources. Storage and expiration dates are listed on the container.
Secondary Antibodies
Secondary Antibodies............................450 ul per individual slide well.
Secondary Antibodies are purchased from BioTek Solutions, Inc.
Storage conditions and expiration dates are listed on the container.
DAB Reagent (DAB - Three, 3'diaminobenzidine tetrahydrochloride)
DAB Reagent.......................................750 ul per individual slide well.
DAB is purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container(s).
DAB Reagent Preparation
Chemistry
Volume of DAB Desired
#
ml
Mix thereughly before aliguottag
Mark the date an the centainer and stere at 4 degrees C. Use within 5 days.
Avidin-Biotin-Alkaline Phosphatase complex Reagent (AP)
AP Reagent....................................325 pil (or 8 drops) per individual slide well
AP is purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container(s).
AP Reagent Preparation
Volume of AP Desired
ABC-AP Reagent “A.”
5 drops
10 drops
12 drops
ABC-AP Reagent "B"
5 drops
12 drops
Mix thoroughly and let incubate for at least 30 minutes before aliquoting.
Date container and store at 4 degrees C. Use within 5 days.
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BT Red Reagent (CHROM GEN) Prepare just before use: Approximately 15 minutes before the
slides reach the chromogen position.
BT Red Reagent.....................................750 ul per individual slide well.
BT Red is purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container(s). This chromogen is fluorescent, as
well as producing an absorbing (visible) product. This chromogen may also be dehydrated
prior to coverslipping. If aqueous media are used for coverslipping, rehydrate the slides
from the final 100% alcohol step prior to using the aqueous mounting medium. Failure to
include the alcohol step may result in the appearance of crystals over the chromogen
deposits.
If the specimen contains endogenous alkaline phophatase, it may be necessary to block this prior to
chromogen development. This may be done by including Levamisole in the BT Red chromogen
(Levamisole is included as a component in the BT RED chromogen kit). Use Levamisole only if
required. For those specimens that do require blocking of endogenous alkaline phosphatase, add one
drop of Levamisole to each 10 ml of Tris buffer used to compound BT Red reagent. Add and
mix before adding BT Red components. After mixing, discard any volume that will not be used
BT RED Reagent Preparation (mix thoroughly after the addition of each reagent)
Chemistry
Volume of BT Red Reagent Required
ism
750 pil
TRIS Buffer
750 pil
27.5m
isºna sm ºn 222.5 ml
| mºnº | ºnlon Bolo, ºn
BT RED #2 (add & mix)
ºn Eon low on
Mix thoroughly before aliquoting.
Use within 15 minutes.
Using the value of 750 pil of reagent per slide pair, 1 slide pair will require 750 ul of reagent; 2 slide
pairs will require 1.5 ml, 10 slide pairs will require 7.5 ml, 20 slide pairs will require 15 ml. A full
slide carrier of 30 slide pairs will require 22.5 ml.
BioTek Hematoxylin (STN)
BT Hematoxylin......................................... 10 mls per 10 well tray
This stain is commercially purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container.
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Procedure
Use Protocol MIP-AP and 2MIP-AP on TechMatc".
Prior to starting TechMate, slides must be organized for the staining run, deparaffinized, hydrated to
buffer (water), and subjected to microwaving in BioTek Microwaving Buffer. Detailed instructions
for these steps are given in the general microwaving procedures. It is critically important to follow
the microwaving procedures precisely, in order to insure reproducible retrieval of antigenic sites. At
the conclusion of the microwaving step, slides must be cooled to room temperature. During this cool
down interval, the TechMate can be prepared for the staining run.
Load reagents into disposable dishes on TechMate according to reagent template and antibody posi
tioning sheet. Take care to load proper reagents and volumes for each reagent. Use particular
caution when loading primary reagents. It is critical that each slide pair receive each reagent step.
Refer to general instructions for additional information on organizing your immunoperoxidase stain
ing run.
Quality Assurance
All staining runs should include known positive control specimens to validate reagent integrity and
proper performance of the sequential staining steps. When stain results are qualitatively or quantita
tively discordant with expected findings, based on hematoxylin and eosinslides, cytologic findings, or
other clinical data, further evaluation will be performed to resolve any discrepancy at the discretion of
the pathologist, and this will be documented.
Results
Epitopes(antigenic sites) recognized by the first primary antibody will be stained a brown color, and
the particular epitopes recognized by the second primary antibody will be stained a red color. The
density of both the red and brown color is directly related to the amount of specific antigen present,
when the stain is performed under controlled conditions, as on the TechMate. Nuclei will have a blue
hematoxylin counterstain.
References
Brigati, D., L.R. Budgeon, E.R. Unger, et al., Immunocytochemistry is Automated: Development of
a Robotic Workstation based Upon the Capillary Action Principle. J. Histotech. 11: 165-183,
1988.
DeLellis, R.A., et al., immunoperoxidase Techniques in Diagnostic Pathology; Report of a Workshop
by the National Cancer Institute. Amer. J. Clin. Path. 71: 483-488, 1979.
Guesdon, J., et al., The use of Avidin-Biotin interaction in immunoenzymatic techniques. J.
Histochem. Cytochem. 27: 1131-1139, 1979.
Larsson, L-I., Tissue Preparation Methods for Light Microscopic Immunohistochemistry. App.
Immunohistochem. 1:2-16, 1993.
Shi, S.R., M.E. Key and K.L. Kalra, Antigen Retrieval in Formalin-fixed Paraffin-embedded Tissues:
An Enhancement Method for Immunohistochemical Staining Based on Microwave Oven
Heating of Tissue Sections. J. Histochem. Cytochem. 39:741-748, 1991.
Shi, S.R., C. Cote, K.L. Kaira and C.R. Taylor. A Technique for Retrieving Antigens in Formalin
Immunohistochemistry. J.Histochem.Cytochem.40: 787-792, 1992.
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Suurmeijer, A.J., and M.E. Boon. Optimizing Keratin and Vimentin Retrieval in Formalin-fixed,
Paraffin-embedded Tissue with the Use of Heat and Metal Salts. App. Immunohistochem.
1:143-148, 1993.
Taylor, C.R., Immunomicroscopy: A diagnostic tool for the surgical pathologist. W.B. Saunders,
Philadelphia, PA 1986.
MIP-AP and 2MIP-AP Protocol
Step Step Name
BUF2A
PADI
BUFIA
PAD1
BUFIA
PAD1
BLOK
PAD)
BUF1A
PADI
ABIA
PADI
BUF1A
PADI
BUF1A
PAD?
BUFIA
PADI
BUFIA
PAD:
BUF1A
PAD1
AB2
PADI
BUFIA
PAD2
BUF2A
PAD2
HP
PAD2
HP
PAD2
HP
PAD2
BUF2A
PAD2
BUF2A
PAD2
BUF2A
PAD2
ABC
:
PAD2
BUF2A
PAD2
BUF2A
Min Time
Max Time
00:00:10
00:00:29
00:10:00
00:00:29
00:00:10
Ö0:00:29
Ö0:00:10
00:00:45
00:10:00
00:00:29
{}0:10:00
O0:07:00
00:00:29
00:00:10
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:00:10
(X):00:29
O0:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:45
00:25:00
00:00:29
00:00::0
00:00:29
00:00:10
00:00:29
00:02:30
00:00:29
00:00:45
()0:10:00
00:00:29
00:00:10
00:0:0:45
00:25:00
00:00:29
00:00::0
00:00:29
00:10:00
Ö0:00:29
{X}:10:00
00:00:29
00:10:00
00:00:29
00:10:00
{N}:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:10:00
00:00:29
Ö0:02:30
00:00:29
00:02:30
00:02:30
00:00:29
00:00:29
00:02:30
00:02:30
00:03:29
00:00: 10
00:00:29
00:00; 10
00:00:29
00:00:10
(X):00:29
{}0:10:00
00:00:29
00:10:00
00:00:29
Q0: $0:00
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:00:45
00:25:00
00:00:29
00:00: 10
00:00:29
00:00: 10
00:10:00
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MIP-AP and 2MIP-AP Protocol, continued
Step Step Name
Min Time
Max Time
Y/N/H
99
100
101
102
103
104
105
106
107
BUF2B
PAD5
BUF3B
PAD5
BUF3B
PAD5
BUF3B
PAD5
BUF3B
{}0:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:10
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00:10:00
{)0:00:29
00:10:00
Yes
Ycs
Yes
Yes
Yes
Yes
Yes
Yes
Ye?
108
109
PAD5
CHROM GEN
00:00:45
00:07:00
00:00:45
00:07:00
Ye?
Yes
1 10
|||
1 12
i 13
114
I 15
i 16
117
! 18
00:00:29
00:00:10
O0:00:45
00:07:00
oo:00:29
00:00:10
00:00:45
00:07:00
00:00:29
00:00:10
00:00:29
00:00:29
00:05:00
00:00:45
00:07:00
00:00:29
00:05:00
Ö0:00:45
00:07:00
00:00:29
| 19
120
PAD5
BUF3B
PAD5
CHROM GEN
PAd5
BUF3B
PAD5
CHROM GEN
PAD5
BUF3B
PAD6
Yes
Ye?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
12I
122
123
BUF3B
PAD6
STN
00:00:10
00:00:29
00:01:00
124
125
126
127
128
i29
130
13|
132
133
134
135
136
PAD6
BUF3B
PAD6
BUF3B
PAO6
H2O
PAD6
H2O
PAD6
i00%
PAD6
100%
PAD6
00:00:29
00:00:10
00:00:29
00:01:00
00:00:29
00:01:00
00:00:29
00:00: 10
00:00:29
00:00, 10
00:00:20
O0:00: 10
O0:00:20
137
100%
00:00. 10
00:10:00
Yes,
t38
PAD6
00:00:20
00:00:20
Yes
00:10:00
00:00:29
00:10:00
00:00:29
00:01:00
00:00:29
00:00:10
00:00:29
00: 10:00
00:00:29
00:10:00
00:00:29
00: 10:00
00:00:29
()0: 10:00
O0:00:20
O0:10:00
00:00:20
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Ye?
Yes
Yes
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TEMPLATE FOR PROTOCOL
MIP-AP
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tutions #g
TEMPLATE FOR PROTOCOL
2MIP-AP
* These positions cannot be used.
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[-,
Page 1
º........ [Adopted
opt TT
PRELIMDNARY DOCUMENTATION
RELEASE
Reviewed
|Reviewed
Reviewed
|
|
|
T.
|
|
|
|
[Reviewed TTT
Under Revision ITT
MIP and 2MIP PROTOCOLS
1. Protocol MP for 60 Place Slide Holder
2. Protocol 2MP for 20 Place Slide Holder
Microwave Antigen Retrieval and Standard
Immunoperoxidase Staining Using
Avidin-Biotin Complex Detection System
TechMate
1. For 60 Place Slide Holder: Follow instructions through the Results section (Page 5). Use the
MIP Protocol (Pages 6-7) and MIP Template (Page 8). Run Protocol MIP which requires a total
time of 2 hours, 33 minutes and 54 seconds and has a total of 82 steps.
2: For 20 Place Slide Holder. Follow instructions through the Results section (Page 5). Use the
2MIPProtocol (Pages 6-7) and 2MIP Template (Page 9). Run Protocol 2MIP (time required is as
noted in #1 above).
(Note: Protocols are identical-only the templates differ)
Principle
The MIP and 2 MIP protocols utilize a type of immunostaining referred to as a “sandwich" tech
nique, in which a specimen is first reacted with an unlabeled primary antibody. Due to the specificity
of the antibody for the desired antigens (epitopes), the primary antibody will react with these anti
gens, forming an antigen-antibody complex. In the second functional step of the protocol, a second
ary antibody directed against the species of the primary antibody is applied to the specimen. This
secondary antibody is biotinylated, that is, the antibody contains one or more biotin molecules that
have been chemically attached to the secondary antibody during its manufacture. The secondary
antibody recognizes any primary antibody that has bound to the specimen. In the third finctional step
of the protocol, a complex of avidin and biotin that is also complexed with peroxidase enzyme (per
oxidase), is permitted to react with the specimen. This complex binds to any sites that have previ
ously reacted with the primary and secondary antibody. The fourth finctional step of the protocol
then provides a chromogenic substrate for the peroxidase of the avidin-biotin-complex. The result of
peroxidase activity on the chromogenic substrate is to render the soluble chromogen into a colored,
insoluble product that precipitates at sites of enzyme activity. This series of functional steps results
5,696,887
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º
2
in the deposition of color at microscopic sites of specimens that contain the specific epitopes recog
nized by the primary antibody. This protocol also requires a pre-treatment of the tissues that consists
of microwaving them in a citrate buffer. This procedure restores the immunoreactivity of many
epitopes that were masked or distorted by tissue fixation treatment.
Specimen
(1) The specimen will consist of tissue sections (paraffin), fixed smears, or cultured cells adhered to
glass slides.
(2) A positive control slide will be run each and every time the stain is performed.
(3) A variety of fixatives may be used for specimen preservation. It is recommended that neutral
buffered formalin be used for 6-12 hours. However, it should be understood that all fixation
procedures result in some reduction of antigen recognition. Formalin fixation should be kept to a
minimum, and the laboratory should test each primary antibody used in a fixation test protocol to
verify that the fixation of the specimen is not creating negative specimens from positive ones.
Specimen Preparation
A. All specimens should be mounted on ChemMate” Capillary Gap Plus glass slides. Speci
mens should be mounted away from the label end of the slide. It is important that specimens be
placed on the side of the slide that holds the painted label area, that is, the “painted” area should be
up. If tissues that have not been mounted on these slides must be stained on the TechMate, pair them
with 130 micron ChemMate" Capillary Gap Plus slides (blank) to create the capillary gap.
ChemMate 130 micron slides have YELLOW painted labels.
B. Paraffin sections should be mounted from a preheated water bath containing distilled or
deionized water. The water bath should contain no additives (such as gelatin, polylysine, etc.).
Sections should be dried by heating, generally at approximately 60°C for a minimum of 60 minutes.
It is important to drain water from beneath the section prior to the oven drying process.
C. Slides containing smears must contain no large clumps or other accumulations of material
that cause “lumpiness” of the smear surface. Any clumps of material will be toothick for adequate
microscopic visualization, and will interfere with capillary gapstaining.
D. Smears or cultured cell preparations should be fixed prior to staining. If the fixative
leaves a residue on the slides, they may require alcohol steps prior to staining. Fixed, air-dried smears
or cultures that do not have a fixative residue (such as carbowax or polyethylene glycol) may be
stained starting with the first aqueous buffer steps of the protocol. It is preferable to prewet the slides
in ChemMate" Buffer #2 in a 10 well tray for several minutes prior to loading into the TechMate"
slide holder. This pre-wetting will ensure adequate fluid flow in the capillary gap.
Preparation of Reagents
Microwaving Buffer
BioTek Microwaving Buffer....................20 mls
Distilled or deionized water.................. 180 mls
This microwave buffer stock is purchased from BioTek Solutions, Inc. For specific
instructions on use of the microwave procedure, refer to the general instructions on
microwaving.
100% Absolute Alcohol
100% Ethanol..........................................25 mls per 10 well tray
This reagent is commercially purchased and stored at room temperature in an approved safety
; cabinet.
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BioTek Solutions, Inc
Buffer Solutions #1, 2 and 3 (BUF1-3)......................................25 mls per 10 well tray
These solutions are commercially purchased from BioTek Solutions, Inc.
Storage conditions and expiration dates are listed on the containers.
Hydrogen Peroxide (HP)
HP Block................................................ 10 mls per 10 well tray
Hydrogen Peroxide Block solution is commercially purchased from BioTek Solutions, Inc.
Storage conditions are listed on the container.
Blocking Antibodies (BLOK)
Blocking Antibody...................................8 drops per individual slide well.
This serum is commercially purchased from BioTek Solutions, Inc.
Primary Antibodies(AB1)
Primary antibody...................................... 8 drops per individual slide well.
Primary antibodies (prediluted) are purchased from BioTek Solutions, Inc., or are
purchased from other sources. Storage and expiration dates are listed on the
container.
Secondary Antibodies (AB2)
Secondary antibodies................................ 8 drops per individual slide well.
Secondary antibody is purchased from BioTek Solutions, Inc. Storage and
expiration dates are listed on the container.
Avidin-Biotin-Enzyme Complex Reagent (ABC)
ABC Reagent.............................…...350 pil (or 8 drops) per individual slide well.
ABC is purchased from BioTek Solutions, Inc. Storage and expiration dates are
listed on the container(s).
ABC Reagent Preparation
Volume of ABC Desired
Mix thoroughly and let incubate for at least 30 minutes before aliquoting.
Date container and store at 4 degrees C. Use within 5 days.
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BioTek Hematoxylin (STN)
BioTek Hematoxylin....................... 10 mls per 10 well tray
This stain is commercially purchased from BioTek Solutions, Inc. Storage conditions are
listed on the container.
DAB Reagent (DAB – Three, 3'diaminobenzidine tetrahydrochloride)
DAB Reagent.............................. 750 pulper individual slide pair well.
DAB is purchased from BioTek Solutions, Inc. Storage and expiration dates are
listed on the container(s).
DAB Reagent Preparation
Chemistry
Volume of DAB Desired
5 ml
ºr
50 ml
60 ml
|x|, |x-lo
"HP for DAB Reagent
TDAB Regent
25 mi
1000 ul | 1200 pil
RoomEoºl lood poºl
Mix thoroughly before aliquoting.
Mark the date on the container and store at 4 degrees C. Use within 5 days.
Procedure
Use Protocols MIP or 2MIP on TechMate". The appropriate template will appear on the screen.
Note: 1) Be sure that 20 Place Slide Holders are placed in their proper HOME positions Row B of any HOME Tile.
2) The 2MIP protocol and template may not be used with a 60 Place Slide Holder.
3) The MIP protocol and template may be used with a 20 place slide holder provided that all
reagents are placed in Row B of their respective tiles and that the slide holder is placed as
noted in 1) above.
Prior to starting TechMate", slides must be organized for the staining run, deparaffinized, hydrated
to buffer (water) and, if desired, subjected to microwaving in ChemMate" Microwaving Buffer.
Detailed instructions for these steps are given in the general microwaving procedures. It is critically
important to follow the microwaving procedures precisely, in order to ensure reproducible retrieval of
antigenic sites. At the conclusion of the microwaving step, slides must be cooled to room tempera
ture. During this cool down interval, the TechMate" can be prepared for the staining run.
Load the MIP or 2MIPprotocol on TechMate". Load reagents into disposable dishes on
TechMate" according to the appropriate reagent template and antibody positioning sheet. Take
care to load proper reagents and volumes for each reagent. Use particular caution when loading
primary reagents. It is critical that each slide pair receive each reagent step. Refer to general instruc
tions for additional information on organizing your immunoperoxidase staining run. After loading
and prior to initiating the run, double check to make sure each reagent has been correctly loaded.
When the run has started, note the time it will end.
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Quality Assurance
All staining runs should include known positive control specimens to validate reagent
integrity and proper performance of the sequential staining steps. When stain results are
qualitatively or quantitatively discordant with expected findings, based on hematoxylin and eosin
slides, cytological findings, or other clinical data, further evaluation will be performed to resolve
any discrepancy at the discretion of the pathologist, and this will be documented.
Results
The particular epitopes (antigenic sites) recognized by the primary antibody will appear brown.
When the stain is performed under controlled conditions, as on the TechMaten...the density of brown
color is directly related to the amount of antigen present. Nuclei will have a blue hematoxylin
counterstain.
References
Brigati, D., L.R. Budgeon, E.R. Unger, et al., Immunocytochemistry is Automated:
Development of a Robotics Workstation based Upon the Capillary Action
Principle. J. Histotech. 11: 165-183, 1988.
DeLellis, R.A., et al., Immunoperoxidase Techniques in Diagnostic Pathology;
Report of a Workshop by the National Cancer Institute. Amer. J. Clin. Path.
71: 483-488, 1979.
Guesdon, J., et al., The use of Avidin-Biotin interaction in immunoenzymatic tech
niques. J. Histochem. Cytochem. 27: 1131-1139, 1979.
Larsson, L-I., Tissue Preparation Methods for Light Microscopic immunohisto
chemistry. App. Immunohistochem. 1:2-16, 1993.
Shi, S.R., M.E. Key and K.L. Kalra, Antigen Retrieval in Formalin-fixed
Paraffin-embedded Tissues: An Enhancement Method for Immunohis
tochemical Staining Based on Microwave Oven Heating of Tissue Sec
tions. J. Histochem. Cytochem. 39:741-748, 1991.
Shi, S.R., C. Cote, K.L. Kalra and C.R. Taylor. A Technique for Retrieving Antigens
in Formalin-fixed, Routinely Acid-decalcified, Celloidin-embedded Human
Temporal Bone Sections for Immunohistochemistry. J. Histochem.
Cytochem. 40: 787-792, 1992.
•
Suurmeijer, A.J.H. and M.E. Boon. Optimizing Keratin and Vimentin Retrieval in
Formalin-fixed, Paraffin-embedded Tissue with the Use of Heat and Metal
Salts. App. Immunohistochem. 1: 143-148, 1993.
Taylor, C.R., Immunomicroscopy: A diagnostic tool for the surgical pathologist.
W. B. Saunders, Philadelphia, PA 1986.
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MIP and 2MIP PROTOCOL
Step
Step Name
Min Time
Max Time
Y/N/H
l
2
3
4
5
6
7
8
9
10
i|
12
BUF2
PAD1
BUFl
PAD i
BUFl
PADI
BLOK
PAD1
BUFl
PAD1
AB1
PADI
00:00:10
00:00:29
00:00: 10
00:00:29
00:00: iO
00:00:45
00:07:00
00:00:29
00:00:10
00:00:45
00:25:00
00:00:29
OO:10:00
00:00:29
00: $0:00
00:00:29
()0; 10:00
00:00:45
00:10:00
00:00:29
00:00: ()
00:00:45
00:25:00
00:00:29
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
13
14
15
16
17
BUFl
PAD1
BUFl
PADI
BUF1
00:00:10
{}0:00:29
00:00:10
00:00:29
00:10:00
00:00:29
00:10:00
Ycs
Yes
Yes
Yes
Ye?
|8
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
PAD1
BUFl
PAdl
00:00:29
00:10:00
00:00:29
Yes
Yes
Yes
00:10:00
00:00:45
{00:25:00
00:00:29
O0:00:10
00:00:29
00:10:00
00:00:29
00:02:30
60:00:29
00:02:30
00:00:29
00:02:03
00:00:29
00:10:00
00:00:29
00:10:00
00:00:29
00: 10:00
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:10:00
00:00:29
Yes
Yes
37
38
29
40
4|
42
43
44
45
46
47
48
49
50
BUF)
PAD2
AB2
PAD2
BUF1
PAD2
BUF2
PAD2
HP
PAD2
HP
PAD2
HP
PAD2
BUF2
PAD2
BUF2
PAD2
BUF2
PAD2
ABC
PAD3
BUF2
PAD3
BUF2
PAD3
BUF3
PAD3
BUF3
PAD3
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00:10
00:00:45
00:25:00
00:00:29
Ö0:00:10
00:00:29
00:00:10
Ö0:00:29
00:02:30
00:00:29
Q0:02:30
(X):00:29
00:02:30
00:00:29
00:00:10
00:00:29
(X}:00:10
00:00:29
00:00:40
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:00: 10
00:00:29
00:00:10
O0:00:29
00:10:00
00:00:29
00:10:00
00:00:29
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Ye?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Ye?
Ye?
Yes
Yes
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MIP and 2MIP PROTOCOL, (continued)
Step
Step Name
Min Time
Max Time
Y/N/H
5:
52
53
54
BUF3
PAD3
DAB
PAD3
00:00:10
00:00:45
00:05:00
00:00:29
00:10:00
00:00:45
00:05:00
00:00:29
Ycs
Yoºs
Yes
Yes
55
56
57
58
59
60
61
62
63
64
DAB
PAD3
DAB
PAD3
BUF3
PAD4
BUF3
PAD4
STN
PAD4
65
66
67
68
69
70
71
72
73
74
75
76
BUF3
PAD4
BUF3
PAD4
BUF2
PAD4
100%
PAD4
100%
PAD4
100%
PAD4
(K):05:00
()0:00:29
00:05:00
00:00:29
00:00:10
00:00:29
00:00:10
00:00:29
00:01:00
00:00:29
00:01:00
00:00:29
00:01:00
00:00:29
00:00:10
00:05:00
OO:00:29
Ö0:05:00
00:00:29
00:10:00
00:00:29
00:10:00
O0:00:29
00:01:00
00:00:29
00:01:00
00:00:29
00:01:00
00:00:29
00:10:00
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
HOME
00:00:29
00:00:29
Yes
00:00:10
00:00:20
Q0:00:10
00:00:20
00:00:10
Ö0:00:20
00:10:00
00:00:20
00:10:00
00:00:20
00:10:00
00:00:20
Yes
Yes
Yes
Yes
Yes
Yes
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TEMPLATE FOR PROTOCOL
2MIP
* These positions cannot be used.
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CONFIDENTIAL:
PRELIMINARY DOCUMENTATION
RELEASE
Adopted
Reviewed
Reviewed
Reviewed
Reviewed
-
|
|
||
||
TT
|
|
Under Revision
MIPE and 2MIPE Protocols
1. Protocol MIPE: For 60 Place Slide Holder
2. Protocol 2MIPE: For 20 Place Siide Holder
Microwave Antigen Retrieval, Enzyme Digestion
and Standard Immunoperoxidase Staining Using
Avidin-Biotin-Complex Detection System
TechMate
1. For 60 Place Slide Holder. Follow all instructions given below through Results section on Page 6.
Use Template MIPE on Page 9 with Protocol MIPE and 2MIPE (Pages 7-8) which requires a total
time of 2 hours, 49 minutes and 45 seconds and has a total of 92 steps.
2. For 20 Place Slide Holder: Follow all instructions given below through Results section on Page 6.
Use Template 2MIPE on Page 10 with Protocol MIPE and 2MIPE (Pages 7-8) requiring a total
time of 2 hours, 49 minutes and 45 seconds and has a total of 92 steps.
(Note: Protocols are identical-only the templates differ)
Principle
The MIPE and 2MIPE protocols utilize a type of immunostaining referred to as a “sandwich" tech
nique, by which a specimen is first reacted with an unlabelled primary antibody. Due to the specificity
of the antibody for the desired antigens (epitopes), the primary antibody will react with these anti
gens, forming an antigen-antibody complex. In the second functional step of the protocol, a second
ary antibody directed against the species of the primary antibody is applied to the specimen. This
secondary antibody is biotinylated, that is, the antibody contains one or more biotin molecules that
have been chemically attached to the secondary antibody during its manufacture. The secondary
antibody recognizes any primary antibody that has bound to the specimen. In the third functional step
of the protocol, a complex of avidin and biotin which is also complexed with peroxidase enzyme
(peroxidase), is permitted to react with the specimen. This complex binds to any sites that have
previously reacted with the primary and secondary antibody. The fourth functional step of the
protocol then provides a chromogenic substrate for the peroxidase enzymes of the avidin-biotin
complex. The result of peroxidase activity on the chromogenic substrate is to render the soluble
chromogen into a colored, insoluble product that precipitates at sites of peroxidase activity. This
series of finctional steps results in the deposition of color at microscopic sites that contain the spe
cific epitopes recognized by the primary antibody.
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The enzyme digestion step in this protocol is used to counteract the deleterious effect of tissue
fixation on some antigenic epitopes. Fixatives, particularly additive fixatives such as formalin, may
cover over antigenic sites or otherwise interfere with access of the primary antibody to the epitopes.
The result of this interference is a reduction or elimination of the ability of the primary antibody to
specifically stain the specimen. By using an enzyme digestion step prior to performing the
immunostaining procedure, some (but not all) of these antigenic sites may be “uncovered” and
become accessible to the primary antibody. In this protocol, a protease enzyme is used to partially
reverse the effects of fixation. This enzyme is chosen because it is capable of restoring some
immunreactivity when used at room temperature. Since the specimen is also subjected to microwave
antigen retrieval in this protocol, only a few epitopes require enzyme treatment, and these with a
reduced enzyme concentration, as compared to specimens not subjected to microwave. It should be
recognized that excessive exposure to enzyme digestion will actually destroy antigenic sites, and
therefore the digestion times specified in this protocol should not be altered.
Specimen
(1) The specimen will consist of tissue sections (paraffin), fixed smears, or cultured cells adhered to
glass slides. (A different protocol is provided for cytospins:CAP).
(2) A positive control slide will be run each and every time the stain is performed.
(3) A variety of fixatives may be used for specimen preservation. However, it should be understood
that all fixation procedures result in some reduction of antigen recognition. Formalin fixation should
be kept to a minimum, and the laboratory should test each primary antibody used in a fixation test to
verify that the fixation of the specimen is not creating negative specimens from positive ones.
Specimen Preparation
(A) All specimens should be mounted on ChemMate" Capillary Gap Plus glass slides.
Specimens should be mounted away from the label end of the slide. It is important that specimens are
placed on the side of the slide that holds the painted label area, that is, the “painted" area should be
up. If tissues that have not been mounted on these slides must be stained on the TechMate, pair them
with 130 micron ChemMate” Capillary Gap slides (blank) to create the capillary gap.
ChemMate" 130 micron slides have YELLOW painted labels.
(B) Paraffin sections should be mounted from a preheated water bath containing distilled or
deionized water. The water bath should contain no additives such as gelatin, polylysine, etc. Sec
tions should be dried by heating, generally at approximately 60°C for a minimum of 60 minutes. It is
important to drain water from beneath the section prior to the oven drying process.
(C) Slides containing smears must contain no large clumps or other accumulations of material
that cause “lumpiness” of the smear surface. Any clumps of material will be too thick for adequate
microscopic visualization, and will interfere with capillary gapstaining.
(D) Smears or cultured cell preparations should be fixed prior to staining. If the fixative
leaves a residue on the slides, they may require alcohol steps prior to staining. Fixed, air-dried smears
or cultures that do not have a fixative residue (such as carbowax or polyethylene glycol) may be
stained starting with the first aqueous buffer steps of the protocol. It is preferable to prewet the slides
in buffer in a Coplinjar prior to loading into the TechMate slide holder. This wetting should be done
with TechMate buffer 2 or 3, for several minutes. This prewetting will ensure adequate fluid flow in
the capillary gap when staining is started in a truncated protocol at an aqueous step
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Preparation of Reagents
100% Absolute Alcohol
100% Ethanol ...............................................25 mls per 10 well tray
This reagent is commercially purchased and stored at room temperature in
an approved safety cabinet.
Buffer Solution Number 1, 2 and 3 (BUF 1-3)
Buffer Solutions............................................25 mls per 10 well tray
These solutions are commercially purchased from BioTek Solutions, Inc.
Storage directions are listed on the container.
Hydrogen Peroxide (HP)
HP Block........................................................ 10 mls per 10 well tray
Hydrogen Peroxide Block solution is commercially purchased from BioTekSolutions, Inc.
Storage conditions are listed on the container.
Enzyme (ENZ)
Enzyme .............…......…........…....500 pil per individual slide pair well.
Enzyme is commercially purchased from BioTek Solutions, Inc.
Storage conditions are listed on the container(s).
Enzyme Preparation
Chemi
Intstry
Volume of Enzyme Desired
sm
2sm
Enzyme Buffer
Concentrated Enzyme
Mix thoroughly and warm to room temperature before aliquoting.
For positions not requiring enzyme, use Enzyme Substitution Buffer.
Blocking Antibodies (BLOK)
Blocking Antibody ........................................8 drops per individual slide pair well.
This serum is commercially purchased from BioTek Solutions, Inc.
Primary Antibodies (AB1)
Primary Antibody.......................................... 8 drops per individual slide pair well.
Primary antibodies (predilute) are purchased from BioTek Solutions, Inc., or from other
sources.
Storage and expiration dates are listed on the container.
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Secondary Antibodies (AB2)
cº,
Secondary Antibodies........................................ 8 drops per individual slide well
Secondary antibody is purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container.
ABC Reagent (ABC Avidin-Biotin-Enzyme Complex)
ABC Reagent........................................….350 ul (or 8 drops) per individual slide well.
ABC is purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container(s).
ABC Reagent Preparation
Chemistry
Volume of ABC Desired
ABC Reagent"B"
Mix thoroughly and let incubate for at least 30 minutes before aliquoting.
Date container and store at 4 degrees C. Use within 5 days.
DAB Reagent (DAB Three, 3'diaminobenzidine tetrahydrochloride)
DAB Reagent................................................750 pul per individual slide well.
DAB is purchased from BioTek Solutions, Inc.
Storage and expiration dates are listed on the container(s).
DAB Reagent Preparation
Volume of DAB Desired
TRIS Buffer
"HP" Reagent
Mix Thoroughly and warm to room temperature before aliquoting.
Mark the date on the container and store at 4 degrees C. Use within 5 days.
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BioTek Hematoxylin
BioTek Hematoxylin............................................ 10 mls per 10 well tray.
This stain is purchased commercially from BioTek Solutions, Inc.
The storage and expiration dates are listed on the container.
Procedure
Load reagents into disposable 10 well trays on TechMate according to reagent template and antibody
positioning sheet. Indicate on antibody positioning sheet those slide pairs requiring enzyme. For
slide pairs not requiring enzyme use enzyme substitution buffer (supplied with the enzyme kit from
BioTek Solutions, Inc.) Indicate these positions on sheet. Take care to load proper reagents and
volumes for each reagent. Use particular caution when loading primary reagents and enzyme. It is
critical that each slide pair receive each reagent step. Refer to general instructions for additional
information on organizing your immunoperoxidase staining run.
Quality Assurance
All staining runs should include known positive control specimens to validate reagent integrity and
proper performance of the sequential staining steps. When stain results are qualitatively or quantita
tively discordant with expected findings, based on hermatoxylin and eosin slides, cytologic findings, or
other clinical data, further evaluation will be performed to resolve any discrepancy at the discretion of
the pathologist, and this will be documented.
Results
Epitopes (antigenic sites) within the specimen that are recognized by the primary antibody will appear
brown. When the stain is performed under controlled conditions, as on the TechMate, the density of
brown color is directly related to the amount of antigen present. Nuclei will have a hematoxyfin blue
counterstain.
References
Brigati, D., L.R. Budgeon, E.R. Unger, et al., Immunocytochemistry Is Automated: Development of
a Robotic Workstation Based Upon the Capillary Action Principle. J. Histotech. 11:165-183.
DeLellis, R.A., et al., Immunoperoxidase Techniques in Diagnostic Pathology; Report of a Work
shop by the National Cancer Institute. Amer. J. Clin. Path. 71; 483-488, 1979.
Guesdon, J., et al., The Use of Avidin-Biotin Interaction in Immunoenzymatic Techniques. J.
Histochem. Cytochem. 27:1131-1139, 1979.
Larsson, L.-J., Tissue Preparation Methods for Light Microscopic Immunohistochemistry. App.
Immunohistochem. 1:2-16, 1993
Shi, S.R., M.E. Key and K.L. Kalra, Antigen Retrieval in Formalin-fixed Paraffin-embedded Tissues:
An Enhancement Method for Immunohistochemical Staining Based on Microwave Oven
Heating of Tissue Sections. J. Histochem. Cytochem. 39:741-748, 1991.
Shi, S.R., C. Cote, K.L. Kalra, anc C.R. Taylor. A Technique for Retrieving Antigens in Formalin
fixed, Routinely Acid-decalcified, Celloidin-embedded Human Temporal Bone Sections for
Immunohistochemistry. J. Histochem. Cytochem, 40: 787-792, 1992.
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Page 6
Shi, S.R., C. Cote, K.L. Kalra, anc C.R. Taylor. A Technique for Retrieving Antigens in Formalin
fixed, Routinely Acid-decalcified, Celloidin-embedded Human Temporal Bone Sections for
Immunohistochemistry. J. Histochem. Cytochem. 40: 787-792, 1992.
Suurmeijer, A.J.H. and M.E. Boon. Optimizing Keratin and Vimentin Retrieval in Formalin-fixed,
Para??in-embedded Tissue with the Use of Heat and Metal Salts. App. Immunohistochem. 1:
143-148, 1993.
Taylor, C.R., Immunomicroscopy: A diagnostic tool for the surgical pathologist. W.B. Saunders,
Philadelphia, PA 1986.
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†-
MIPE and 2MIPE Protocols
Step Step Name Min Time
Max Time
I
2
3
4
5
6
7
BUF2
PAD1
BUF2
PAD?
BUF2
pAD1
ENZ
00:00:10
00:00:29
()0:00:10
00:00:29
00:00:10
00:00:45
00:10:00
8
9
10
il
#2
13
PAD1
BUF2
PAD?
BUFl
PAD1
BUFl
00:00:29
00:00:10
00:00:29
00:00: 10
00:00:29
00:00:40
14
15
16
17
18
PAD1
BUF1
PADI
BLOK
PAD1
00:00:29
00:00:10
00:00:45
00:07:00
00:00:29
19
20
21
22
BUF1
PAD1
AB1
PAD1
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
BUFi
PAD2
BUF1
PAD2
BUF1
PAD2
BUF1
PAD2
BUFl
PAD2
AB2
PAD2
BUF
PAD2
BUF2
pAD2
HP
PAD2
00:00:10
00:00:45
00:25:00
00:00:29
00:00:10
00:10:00
00:00:29
00: 10:00
00:{x}:29
(x): [0:00
00:00:45
00:10:00
00:00:29
00:09:10
(x0:00:29
00:10:00
00:00:29
O0:10:00
00:00:29
00:10:00
00:00:45
00:10:00
O0:00:29
00:00:10
41
42
43
44
HP
PAD2
HP
PAD2
45
46
47
48
49
BUF2
PAD3
BUF3
PAD3
BUF3
50
PAD3
00:00:29
00:09:10
00:00:29
00:00; iO
00:00:29
00:00:10
O0:00:29
00:00:10
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:00:40
00:00:29
00:02:30
O0:00:29
00:02:30
O0:00:29
00:02:30
00:00:29
00:00:10
00:0C:29
00:00:40
00:00:29
00:00:10
00:00:45
00:00:45
00:25:00
00:00:29
00:00:10
00:00:29
00:10:00
00:00:29
Q0:10:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:45
?}0:25:00
00:00:29
00:00: 10
?0:00:29
00:10:00
00:00:29
00:02:30
00:00:29
00:02:30
00:00:29
00:02:30
00:00:29
{}0: 0:00
00:00:29
00:10:00
00:00:29
00:10:00
00:00:45
Yes
Ye?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
. Yes
Yes
Yes
Yes
Yes
Yes
Ye?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Ye?
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
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MIPE and 2MIPE Protocols, continued
Step Step Name Min Time
5t
52
53
54
55
56
57
58
59
60
6]
62
63
64
65
66
67
68
69
70
7t
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
9|
92
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PAD3.
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PAD3
BUF3
PAD3
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BUF3
PAD4
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PAD3
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PAD3
pAB
PAD3
BUF2
PAD3
DAB
Max Time
00:25:00
00:00:29
00:25:00
00:00:29
00:00:10
00:00: 10
00:00:29
00:00:10
00:00:29
{X):00:10
{X):00:29
00:00:10
00:00:29
00:00:10
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00:00:29
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MIPE
|-|--|->|
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= **E* 10
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TEMPLATE FOR PROTOCOL
2MIPE
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CONFIDENTIAL:
|Adopted
|
PRELIMINARY DOCUMENTATION | Reviewed
RELEASE
Reviewed ºf
Reviewed
TReviewed TTT
Under Revision
MAPE and 2MAPE Protocols
1. Protocol MAPE: For 60 Place Slide Holder
2. Protocol 2MAPE: For 20 Place Slide Holder
Microwave Antigen Retrieval, Enzyme Digestion, and Standard
Immuno-Alkaline Phosphatase Staining Using Alkaline
Phosphatase in an Avidin-Biotin-Complex
Detection System
TechMate
1. For 60 Place Slide Holder. Follow all instructions given below through Results section on Page 6.
Use Template MAPE on Page 9 with Protocol MAPE and 2MAPE (Pages7-8) which requires a
total time of 2 hours, 45 minutes and 18 seconds and has a total of 86 steps.
2. For 20 Place Slide Holder. Follow all instructions given below through Resuits section on Page 6.
Use Template 2MAPE on Page 10 with Protocol MAPE and 2MAPE (Pages 7-8) requiring a total
time of 2 hours, 45 minutes and 18 seconds and has a total of 86 steps.
(Note: Protocols are identical-only the templates differ)
Principle
The MAPE and 2MAPE protocol utilizes a type of immunostaining referred to as a “sandwich"
technique, in which a specimen is first reacted with an unlabelled primary antibody. Due to the
specificity of the antibody for the desired antigens (epitopes), the primary antibody will react with
these antigens, forming an antigen-antibody complex. In the second functional step of the protocol, a
secondary antibody directed against the species of the primary antibody is applied to the specimen.
This secondary antibody is biotinylated, that is, the antibody contains one or more biotin molecules
that have been chemically attached to the secondary antibody during its manufacture. The secondary
antibody recognizes any primary antibody that has bound to the specimen. In the third functional step
of the protocol, a complex of avidin and biotin that is also complexed with alkaline phosphatase
enzyme (alkaline phosphatase) is permitted to react with the specimen. This complex binds to any
sites that have previously reacted with the primary and secondary antibody. The fourth functional
step of the protocol then provides a chromogenic substrate for the alkaline phosphatases of the
avidin-biotin-complex. The result of alkaline phophatase activity on the chromogenic substrate is to
render the soluble chromogen into a colored, insoluble product that precipitates at sites of alkaline
phophatase activity. This series of functional steps results in deposition of color at microscopic sites
of specimens which contain the specific epitopes recognized by the primary antibody.
The enzyme digestion step in this protocol is used to counteract the deleterious effect of tissue fixa
tion on some antigenic epitopes. Fixatives, particularly additive fixatives such as formalin, may cover
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over antigenic sites or otherwise interfere with access of the primary antibody to anugenic epitopes.
The result of this interference is a reduction or elimination of the ability of the primary antibody to
specifically stain the specimen. By using an enzyme digestion step prior to performing the
immunostaining procedure, some (but not all) of these antigenic sites may be “uncovered” and be
corne accessible to the primary antibody. In this protocol, a protease enzyme is used to partially
reverse the effects of fixation. This enzyme is chosen because it is capable of restoring some immu
noreactivity when used at room temperature. Since the specimen is also subjected to microwave
antigen retrieval in this protocol, only a few epitopes require enzyme treatment, and these with a
reduced enzyme concentration, as compared to specimens not subjected to microwave. It should be
recognized that excessive exposure to enzyme digestion will actually destroy antigenic sites, and
therefore the digestion times specified in this protocol should not be altered.
Specimen
(1) The specimen will consist of tissue sections (paraffin), fixed smears, or cultured cells adhered to
glass slides (a different protocol is provided for cytospins: CAP).
(2) A positive control slide will be run each and every time the stain is performed.
(3) A variety of fixatives may be used for specimen preservation. However, it should be understood
that all fixation procedures result in some reduction of antigen recognition. Formalin fixation should
be kept to a minimum, and the laboratory should test each primary antibody used in a fixation test
protocol to verify that the fixation of the specimen is not creating negative specimens from positive
oncs.
Specimen Preparation
A. All specimens should be mounted on ChemMate"Capillary Gap glass slides. Specimens
should be mounted away from the label end of the slide. It is important that specimens be placed on
the side of the slide that holds the painted label area, that is, the "painted area" should be up. If
tissues that have not been mounted on such slides must be stained on the TechMate, pair them with
ChemMate” 130 micron yellow labeled slides (blank) to create the capillary gap. ChemMate"
130 micron slides have YELLOW painted labels.
B. Paraffin sections should be mounted from a preheated water bath containing distilled or
deionized water. The water bath should contain no additives such as gelatin, polylysine, etc. Sec
tions should be dried by heating, generally at approximately 60°C for a minimum of 60 minutes. It is
important to drain water ?tom beneath the section prior to the oven drying process.
C. Slides containing smears must contain no large clumps or other accumulations of material
that cause “lumpiness” of the smear surface. Any clumps of material will be toothick for adequate
microscopic visualization, and will interfere with capillary gap staining.
D. Smears or cultured cell preparations should be fixed prior to staining. If the fixative
leaves a residue on the slides, they may require alcohol steps prior to staining. Fixed, air-dried smears
or cultures that do not have a fixative residue (such as carbowax or polyethylene glycol) may be
stained starting with the first aqueous buffer steps of the protocol. It is preferable to prewet the slides
in buffer in a Coplin jar prior to loading into the TechMate slide holder. This wetting should be done
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with TechMate buffer 2 or 3, for several minutes. This pre-wetting will ensure an adequate ?luid
flow in the capillary gap when staining is started in a truncated protocol at an aqueous step.
Preparation of Reagents
Microwaving Buffer
BioTek Microwaving Buffer....................25 mls per 10 well tray
This microwave buffer stock is purchased from BioTek Solutions, Inc. For specific instruc
tions on use of the microwave procedure, refer to the general instructions on microwaving.
100% Absolute Alcohol
100% Ethanol..........................................25 mls per 10 ml tray
This reagent is commercially purchased and stored at room temperature in an
approved safety cabinet.
Buffer Solutions Numbers 1,2 and 3 (BUF 1-3)
Buffer Solutions ......................................25 mls per 10 well tray
These solutions are commercially purchased from BioTek Solutions, Inc.
Storage directions are listed on the container.
Blocking Antibodies (BLOK)
Blocking Antibody...............................8 drops perindividual slide well.
This serum is commercially purchased from BioTek Solutions, Inc.
Enzyme (ENZ)
Enzyme.....................-------------------. .500 pil per individual slide well
Enzyme is commercially purchased from BioTek Solutions, Inc.
Storage conditions are listed on the container(s).
Enzyme Preparation
Chemistry
Volume of Enzyme Desired
Enzyme Buffer
Mix thoroughly and warm to room temperature before aliquoting.
For positions not requiring enzyme, use Enzyme Substitution Buffer.
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Primary Antibodies (AB1)
Primary antibody.....................----------.8 drops per individual slide well. Primary
antibodies (predilute) are purchased from BioTek Solutions, Inc., or are purchased from other
sources. Storage and expiration dates are listed on the container.
Secondary Antibodies (AB2)
Secondary antibodies..........................8 drops per individual slide well. Secondary
antibody is purchased from BioTek Solutions, Inc. Storage and expiration dates are listed on
the container.
Avidin-Biotin-Alkaline Phosphatase Complex Reagent (AP)
AP Reagent.......................................... 325 ul (or 8 drops) per individual slide well
AP is purchased from BioTek Solutions, Inc. Storage and expiration dates are listed on the
container(s).
AP Preparation
Chemistry
Volume of AP Desired
sm
rºsºs | sm
5 ml
AP-ABC Reagent "A"
sm sºn I am
ºn | Som
25 ml
5 drops
AP-ABC Reagent "B"
10 drops
12 drops
Mix thoroughly and let incubate for at least 30 minutes before aliquoting.
Date container and store at 4 degrees C. Use within 5 days.
BioTek Hematoxylin (STN)
BioTek Hematoxylin................................. 10 misper 10 well tray
This stain is commercially purchased from BioTek Solutions, Inc. Storage conditions are
listed on the container.
BT Red Reagent (a New Fuchsin-type chromogen)
Prepare just before use! Approximately 15 minutes before the slides reach the
chromogen position.
BT Red Reagent......................................750 ul per individual slide pair well.
BT Red is purchased from BioTek Solutions, Inc. Storage and expiration dates are listed on
the container(s). This chromogen is fluorescent, as well as producing an absorbing (visible)
product. This chromogen may also be dehydrated prior to coverslipping. I?aqueous media
are used for coverslipping, rehydrate the slides from the final 100% alcohol step prior to us
ing the aqueous mounting medium. Failure to include the alcohol step may result in the
appearance of crystals over the chromogen deposits.
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If the specimen contains endogenous alkaline phosphatase, it may be necessary to block this prior to
chromogen development. This may be done by including Levamisole in the the BT Red chromogen
(Levamisole is included as a component in the BT Red chromogen kit). Use Levamisole only if
required. For those speciments that do require blocking of endogenous alkaline phosphatase, add
one drop of Levamisole to each 10 ml of Tris Buffer used to compound BT Red reagent. Add
and mix before adding BT Red components. After mixing, discard any volume that will not be
used for making BT Red reagent.
BT Red Reagent Preparation (mix thoroughly after the addition of each reagent)
Chemistry
Volume of BT Red Desired
Tsºul IIsm. I sº I isºl Esm
TRIS Buffer
BT Red #1 (add & mix}
BT Red #3 (add & mix)
Mix thoroughly before aliquoting. Use within 15 minutes.
Using the value of 750 pil of reagent per slide pair, 1 slide pair will require 750 ul of reagent; 2 slide
pairs will require 1.5 ml, 10 slide pairs will require 7.5 ml, 20 slide pairs will require 15 ml. A full
slide carrier of 30 slide pairs will require 22.5 ml.
Procedure
Use protocol MAPE AND 2MAPE on TechMate.
Prior to starting TechMate, slides must be organized for the staining run, deparaffinized, hydrated to
buffer (water), and subjected to microwaving in BioTek Microwaving Buffer. Detailed instructions
for these steps are given in the general microwaving procedures. It is critically improtant to follow
the microwaving procedures precisely in order to ensure reproducible retrieval of antigenic sites. At
the conclusion of the microwaving step, slides must be cooled to room temperature. During this cool
down interval, the TechMate can be prepared for the staining run
Quality Assurance
All staining runs should include known positive control specimens to validate reagent integrity and
proper performance of the sequential staining steps. When stain results are qualitatively or quantita
tively discordant with expected findings, based on hematoxylin and eosin slides, cytologic findings, or
other clinical data, further evaluation will be performed to resolve any discrepancy at the discretion of
the pathologist, and this will be documented.
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Results
Epitopes (antigenic sites) recognized by the primary antibody will appear RED. The density of red
color is directly related to the amount of antigen present, when the stain is performed under con
trolled conditions, as on the TechMate. Nuclei will have a hematoxylin blue counterstain.
References
Brigati, D., L.R. Budgeon, E.R. Unger, et al., Immunocytochemistry is Automated: Development of
a Robotic Workstation based Upon the Capillary Action Principle. J. Histotech. 11: 165-183,
1988.
DeLellis, R.A., et al., Immunoperoxidase Techniques in Diagnostic Pathology; Report of a Workshop
by the National Cancer Institute. Amer. J. Clin. Path. 71:483-488, 1979.
Guesdon, J., et al., The use of Avidin-Biotin interaction in immunoenzymatic techniques. J.
Histochem. Cytochem. 27: 1131-1139, 1979.
Larsson, L-I., Tissue Preparation Methods for Light Microscopic Immunohistochemistry. App.
Immunohistochem. 1:2-16, 1993.
Shi, S.R., M.E. Key and K.L. Kalra, Antigen Retrieval in Formalin-fixed Paraffin-embedded Tissues:
An Enhancement Method for Immunohistochemical Staining Based on Microwave Oven
Heating of Tissue Sections. J. Histochem. Cytochem. 39:741-748, 1991.
Shi, S.R., C. Cote, K.L. Kaira and C.R. Taylor. A Technique for Retrieving Antigens in Formalin
fixed, Routinely Acid-decalcified, Celloidin-embedded Human Temporal Bone Sections for
Immunohistochemistry. J. Histochem. Cytochem. 40: 787-792, 1992.
Suurmeijer, A.J.H. and M.E. Boon. Optimizing Keratin and Vimentin Retrieval in Formalin-fixed,
Paraffin-embedded Tissue with the Use of Heat and Metal Salts. App. immunohistochem. 1:
143-148, 1993.
Taylor, C.R., Immunomicroscopy: A diagnostic tool for the surgical pathologist. W. B. Saunders,
Philadelphia, PA 1986.
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MAPE and 2MAPE Protocol, continued
Step
Step Name
Min Time
Max Time
Y/N/H
5|
52
BUF3
PAD3
00:00:10
00:00:29
00:10:00
00:00:29
Yes
Yes
53
54
55
56
57
58
59
60
6|
62
63
64
BUF3
PAD3
BUF3
PAD3
CHROM GEN
PAD3
BUF3
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CHROM GEN
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00:00:10
00:00:29
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00:07:00
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00:07:00
00:00:29
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65
66
CHROM GEN 00:07:00
PAD3
00:00:29
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67
68
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00:00:10
00:00:29
69
BUF3
00:00:10
70
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00:00:29
71.
STN
00:01:00
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00:01:00
72
73
74
75
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pAdA
BUF2
00:00:29
00:00:10
00:00:29
00:01:00
76
77
78
79
80
8}
82
83
84
85
86
PAD4
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PAD4
H2O
PAD4
100%
PAO.4
100%
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100%
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#
PAD1
BUF2
BUF3
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PAD2
100%
PAD3
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