LR White embedding kit Instructions &

Distributed By:
Ladd Research
83 Holly Court
Williston, VT 05495 USA
Telephone: (802) 658-4961
Fax: (802) 660-8859
Email: sales@laddresearch.com
LR White Embedding Kits - 12390 and 12392
I. Introduction ...........................................................................................................................................................2
II. Applications .........................................................................................................................................................2
1. LR White for Electron Microscopy...................................................................................................................2
1.1 Fixation......................................................................................................................................................2
1.2 Dehydration...............................................................................................................................................2
1.3 Infiltration...................................................................................................................................................2
1.4 Prepolymerisation .....................................................................................................................................2
1.5 Polymerisation ..........................................................................................................................................2
1.6 Trimming and Cutting................................................................................................................................3
1.7 Section Staining ........................................................................................................................................3
1.8 In the Electron Microscope .......................................................................................................................3
2. LR White for Electron Microscopic Immunocytochemistry..............................................................................4
2.1 Introduction ...............................................................................................................................................4
2.2 Fixation......................................................................................................................................................4
2.3 Dehydration...............................................................................................................................................4
2.4 Infiltration...................................................................................................................................................5
2.5 Prepolymerisation .....................................................................................................................................5
2.6 Polymerisation ..........................................................................................................................................5
2.7 Staining .....................................................................................................................................................5
3. LR White for Light Microscopy ........................................................................................................................6
3.1 Introduction ...............................................................................................................................................6
3.2 Fixation......................................................................................................................................................6
3.3 Dehydration...............................................................................................................................................6
3.4 Infiltration...................................................................................................................................................6
3.5 Prepolymerisation .....................................................................................................................................6
3.6 Polymerisation ..........................................................................................................................................6
3.7 Cutting and Mounting................................................................................................................................7
3.8 Section Staining ........................................................................................................................................7
4. LR White for Immunohistochemistry ...............................................................................................................8
4.1 Introduction ...............................................................................................................................................8
4.2 PAP Procedure for LR White Sections (3µm)...........................................................................................9
4.1 General Staining Procedure – Indirect Technique....................................................................................9
4.2 Antigen Retrievals .....................................................................................................................................9
5. LR White for Hard Tissue ..............................................................................................................................10
5.1 Introduction .............................................................................................................................................10
5.2 Decalcified Tissue...................................................................................................................................10
5.3 Non-decalcified Tissue............................................................................................................................10
5.4 Dehydration.............................................................................................................................................10
5.5 Infiltration.................................................................................................................................................10
5.6 Prepolymerisation ...................................................................................................................................10
5.7 Polymerisation ........................................................................................................................................10
5.8 Cutting and Mounting..............................................................................................................................10
5.9 Microtomy................................................................................................................................................10
5.10 Sawing and Grinding.............................................................................................................................11
5.11 Section Staining ....................................................................................................................................11
III. Ordering information .........................................................................................................................................11
1
I. Introduction
LR White Resin is a very low viscosity resin, non-toxic and suitable for various applications in both light and
Electron Microscopy.
Major advantage of LR White sections is that they show minimal non-specific staining. Sections of polymerized
resin are hydrophilic, thus immunocytochemistry reagents easily penetrate into the section.Its low viscosity
makes LR White an ideal tool for infiltrating plant tissues as well as decalcified bone and teeth.
II. Applications
1. LR White for Electron Microscopy
When using LR White embedding resin for electron microscopy, very few changes need to be made to the
regime used for epoxy resin embedding. Every laboratory has its own individual embedding methods and we
have described here a typical method for LR White as guidance for its use.
1.1 Fixation
No change from normal fixation should be made, if only EM is required from the final blocks.
If, however, good ultrastructure and a wide range of LM staining is required then we have found that the use of
freshly depolymerised paraformaldehyde (3-4%) in a phosphate buffer pH 7,2 with 2,5% w/v sucrose (Fluka
840999) is the best compromise. Glutaraldehyde (Ladd #20100) alone and Karnovsky’s glutaraldehydeformaldehyde mixtures may lead to patchiness. LM staining and some stains which are not functioning or giving
“false positives“ (e.g. PAS) whereas normal formalin fixation yields unacceptable EM ultrastructure.
For the dual LM/EM role osmium tetroxide should be avoided due to its effect on many LM stains but 1% (w/v)
phosphotungstic acid (Ladd #23610) in the first absolute alcohol step of dehydration improves electron contrast
without adversely affecting most LM stains. Osmium tetroxide (Ladd # 20280) may be used if the blocks are
required for EM only.
1.2 Dehydration
A graded ethanol series is the method of choice when embedding in LR White. Acetone acts as a radical
scavenger in the resin system and therefore traces of acetone left in the tissue during curing can interfere with
polymerisation. For this reason the use of a graded acetone series and 2,2-dimethoxypropane, which generates
acetone, are best avoided. If the use of 2,2-dimethoxypropane (Ladd #20410) is considered vital we recommend
either a protracted resin infiltration or washing the tissue with dry ethanol prior to infiltration in order to minimize
the risk of acetone contamination of the final resin.
1.3 Infiltration
The extreme low viscosity of LR White may be exploited by allowing the use of short infiltration times or large
specimens BUT NOT BOTH! A 1mm cube of animal tissue will be adequately infiltrated in about 3 hours if 4-6
0
changes of LR White at 60 C are employed during this period. An overnight infiltration at room temperature,
followed by two short changes of resin will often be more convenient, however. The long shelf life and low
extraction rate of LR White allows specimens (perhaps reserve tissue) to be stored safely in resin for many
weeks at 4°C if required. Larger blocks do require significantly longer infiltration times than small ones.
1.4 Prepolymerisation
The LR White catalyst is a form of benzoyl peroxide in a solid solution to render it safe for transport. One 500 ml
bottle of LR White resin requires 9,9 g of catalyst to be added. The catalyst should be added to the resin at
room temperature and the resin must be shaken thoroughly immediately after addition of catalyst. The catalyst
will take a full 24 hours at room temperature to dissolve completely and during this time it is most helpful if the
bottle can be shaken from time to time. Do not attempt to heat the resin in order to speed the dissolution of the
catalyst.
Once mixed and fully dissolved the shelf life of the catalyzed resin stored at 4°C is at least 12 months.
1.5 Polymerisation
Osmium tetroxide (Ladd #20275 & 20280) reacted tissue should not be “cold-cured“ with the accelerator. This
process is strongly exothermic and the dark colour of the tissue leads to a focal heat accumulation which can
cause local problems in and around the tissue.
2
If the tissue is not osmium tetroxide post-fixed, curing with LR White accelerator may be employed. As with
curing blocks for LM we recommend cooling the moulds during polymerisation, but there is no need to exclude
oxygen from the surface of the curing block.
Thermal curing should be used for osmium treated specimens and may be used for all specimens.. Here it is
important to limit the contact of oxygen with the resin while polymerisation occurs. The most convenient way of
achieving this with capsule-type embedding is to use gelatin capsules, fill up to the brim and slide the other half
of the capsule on.
If flat embedding is required for cutting orientation then the surface of the resin must be covered to prevent
contact with oxygen. One convenient method is to utilize the JB-4-type moulds
and chucks, useful for LM. After polymerisation the block can be sawed off the stub and
mould re-used.
Polymerisation time and temperature are fundamental to the physical characteristics of the final block, to a
°
°
much higher degree than with undercured epoxy systems. We strongly recommend a temperature of 60 ± 2 C
for a period of 20-24 hours.
Some ovens are not capable of controlling polymerisation temperature so closely, and if faced with over brittle
blocks, this is the first parameter to check.
LR White has extremely good powers of penetration and can penetrate and soften some low-density
polyethylene capsules. This causes them to distort and collapse. Also polyethylene is not impermeable to
oxygen and may allow enough contact with atmospheric oxygen to give the blocks an inhibited “tacky“ surface.
Both these problems may be overcome by the use of gelatin capsules (size 00 is similar to the popular
polyethylene capsule size) and these are much cheaper and easier to seal during polymerisation.
The Resin may be used straight from the refrigerator and has a very low toxicity both in the monomeric and
polymerized state, unlike epoxies (see Proc. Roy. Mic. Soc. 16, Pt. 4 p. 265-271). The cold cure accelerator
does have some toxic risk and contact with skin and eyes should be avoided.
For cold curing the accelerator should be used at one drop per 10 ml of resin and this should cause
polymerisation within 10 and 20 minutes. If polymerisation occurs faster than this we recommend either more
careful metering of the one drop of accelerator or a higher volume of resin per drop of accelerator.
1.6 Trimming and Cutting
Trimming the block may be done with jewellers saw, razor blade or a with glass knife on the ultramicrotome, as
with epoxy blocks.
Cutting, too, may be performed in the same way as for epoxy resin with glass or diamond knives. A typical
cutting speed of about 1 mm per second is suitable.
1.7 Section Staining
All the common section stains give good results with tissue embedded in LR White resin. Stains made up in
ethanol or methanol should be avoided as these solvents soften the resin and may remove sections from grids.
As a alternative to uranyl acetate (Fluka 94260), 1% phosphotungstic acid has proved to be a
good general purpose stain, both as a block stain, as mentioned earlier, and as a section stain.
1.8 In the Electron Microscope
An initial reduction in electron density may accompany the initial exposure of the resin to the beam. This is
thought to represent a loss of water, from knife-boat or staining solution. Thinning as such does not occur and
specimens have been kept stationar under a 120 kV electron beam for 3 hours with no obvious sign of damage.
3
2. LR White for Electron Microscopic Immunocytochemistry
2.1 Introduction
LR White resin has five advantages which can be exploited for the localisation of antigens in sections of fixed
and embedded tissue under the electron microscope.
1. It is a hydrophilic embedding agent, which means that ultra-thin sections allow the passage of aqueous
solutions even at neutral pH, as opposed to the epoxides and polyesters, ultra-thin sections of which are
much less permeable.
2. Its lipid solvency is apparently low for a plastic embedding agent and therefore membrane and cytosol
structures can be observed under the electron microscope even when osmium has not been used to
stabilize lipids. No low temperature methods are required although tissue structure is much improved by
perfusion fixation methods.
3. It does not, prevent the demonstration of antigen by immunochemical techniques. No “etching“ or protease
digestion has so far been necessary.
4. It is beam-stable, standing up well to even quite low kV’s thus representing a considerable advance over the
more commonly used methacrylates.
5. It tolerates rapid, partial dehydration, accepting tissue from 70% ethanol. Such tissue has an improved
antigenicity over tissue which has been fully dehydrated.
2.2 Fixation
To preserve antigenicity post-fixation in osmium tetroxide is best avoided but then a
variety of possibilities is available depending on the requirements of the investigator. If tissue blocks are kept
3
small (1-3 mm ) four to six hours fixation in freshly depolymerized and purified 4% paraformaldehyde in a 0,1 M
phosphate buffer pH 7,3 is recommended for the preservation of
maximum tissue antigenicity. Tissue is then washed overnight in buffer solution. Picric acid,
included with formaldehyde, will improve tissue structure slightly and Zamboni’s fixative (Stephanini et al. Nature
216, 1967; 173-174) which is 4% phosphate-buffered paraformaldehyde with picric acid can be used.
Glutaraldehyde, with its greather power to cross-link proteins, undoubtedly stabilises tissue structure to a
greather degree than does formaldehyde, but in turn, tissue thus fixed demonstrates a reduced antigenicity. It is
strongly recommended that the vacuum-distilled, purified form of monomeric or trimeric glutaraldehyde is used,
to increase method reproducibility and avoid deleterious fixative impurities. Three to four hours in 1-2%
glutaraldehyde in 0,1 M phosphate buffer pH 7,3, is perfectly adequate, remembering that the lower the
concentration of glutaraldehyde the higher the antigenicity yield. Tissue is then washed overnight in buffer.
The effect of the avoidance of osmium tetroxide can be partially compensated, without detriment to tissue
antigenicity, by the inclusion of picric acid in the glutaraldehyde solution. The suggested solution is as follows: 2
ml 50% purified glutaraldehyde + 15 ml picric acid (sat.aq.) + 83 ml 0,1 M phosphate buffer pH 7,3 giving a 1%
glutaraldehyde, 0,25% picric acid solution. The percentage of glutaraldehyde and picric acid can be increased to
give a more stable ultrastructure but this may be at the expense of antigenicity. Two to four hours for the fixation
3
of 1-3 mm blocks is recommended. More membrane structure is seen with this fixative than when
glutaraldehyde is used alone,especially if it is perfused into animal tissue, when the molarity of the buffer should
be decreased (0,05-0,08 M).
Further steps, such as post-osmication or “block-staining“ should not be carried out, because they may reduce
immunocytochemical sensitivity – either through reduced antigenicity or enhanced background.
2.3 Dehydration
Tissue fixed in aldehyde alone, after buffer rinses, is dehydrated in a graded ethanol series. Tissue fixed in
aldehyde/picric acid is placed directly into 70% ethanol where some of the picric acid can be washed out.
Reducing the time in ethanol, and the concentration of ethanol used in dehydration will often increase antigenic
yields. LR White will accept tissue from 70% ethanol so that after two washes of 30-60 minutes each, blocks
may be transfered into LR White. When osmium is avoided, tissue shrinkage can be a problem and tissue taken
from fixative in 70% ethanol and then straight into LR White may show shrinkage artefacts. This can be
lessened by introducing an intermediate step of diluted LR White which is 2:1 LR White to 70% ethanol. Be
careful not to carry over large amounts of 70% ethanol when transferring tissue.
4
2.4 Infiltration
Even though the tissue may be taken from 70% ethanol into LR White no special procedures are necessary.
One Change for an hour, followed by an overnight change ( preferably on a “rotamix“) and then a final change
the following morning before embedding, using gelatine capsules, is usually sufficient unless the blocks of tissue
3
°
are particularly large (i.e. in excess of 3 mm ). Blocks may be stored in unpolymerised resin at 4 C for weeks if
necessary.
2.5 Prepolymerisation
The LR White catalyst is a form of benzoyl peroxide in a solid solution to render it safe for transport. One 500 ml
bottle of LR White resin requires 9,9 g of catalyst to be added. The catalyst should be added to the resin at
room temperature and the resin must be shaken thoroughly immediately after addition of catalyst. The catalyst
will take a full 24 hours at room temperature to dissolve completely and during this time it is most helpful if the
bottle can be shaken from time to time. Do not attempt to heat the resin in order to speed the dissolution of the
catalyst.
Once mixed and fully dissolved the shelf life of the catalyzed resin stored at 4°C is at least 12 months.
2.6 Polymerisation
Some special care must be taken to ensure that tissue undergoing embedding is not exposed to temperature in
0
excess of 55 C if antigenicity is not be impaired. The “cold-cure“ procedure (see LR White for Electron
Microscopy), should not be used (the exothermic reaction may exceed 600C) even though the tissue is not
0
ossmium treated, instead an accurate oven or incubator set at 50 C is preferred and a 24 hour polymerisation
time is given. Although anaerobic polymerisation is advised for Beem capsules, in fact LR White polymerises
0
well in a 50 C oven without further precautions when contained in gelatin capsules, fully filled and thightly
capped.
Trimming, cutting and use in the electron microscope are as for the application sheet for electron microscopy.
2.7 Staining
The choice of immunolocalisation technique is entirely up to the user, and PAP, hapten-anti-hapten, avidinbiotin or gold-colloid methods may all be adapted.
Of cource, from time to time, ultra-thin sections of tissue fixed, processed and embedded as described above
should still be stained with uranyl acetate (15 minutes)(Ladd 23620) followed by lead citrate (3 minutes)
(Ladd23610) on the grids as for routine electron microscopy. The
appearance of such tissues is different from that of post-osmicated tissue and it is important that the observer
understands the material with which he is working.
Immunostained sections can be counterstained with lead citrate but if peroxidase techniques are in use this may
confuse the picture.
5
3. LR White for Light Microscopy
3.1 Introduction
Resin embedding for light microscopy provides greatly improved cellular definition compared to paraffin
embedding, and for this reason is now widely used in diagnoses particularly of renal disease, lymphomas and
bone marrow trephines, as well as in research.
The acrylic resins currently used however are not suitable for electron microscopy (EM) and the epoxy resins
used for EM are not easily stained for light microscopy (LM). LR White however can be used for both purposes
and a lymph node for example (12x10x3mm) can be processed, cut and stained for LM, then the same block
trimmed down, cut and stained for EM.
LR White can also be used for the histochemical demonstration of some of the more resistant enzymes, and for
the immunocytochemical demonstration of intracellular immunoglobulins.
For those laboratories already using an acrylic resin e.g. HEMA or GMA, no alteration need be made to the
current processing method, but we have proposed a “typical“ method for LR White as guidance for its use.
3.2 Fixation
No change from normal fixation need be made if LM only is required from the final blocks ( neutral buffered
formalin recommended). If however EM is required subsequent to LM we
have found the use of freshly depolymerised paraformaldehyde (3-4%) in a phosphate buffer pH 7,2 with 2,5%
w/v sucrose is the best compromise. Glutaraldehyde-formaldehyde-mixtures may lead to very pale staining with
haematoxylin and patchy eosin, whereas normal formalin fixation gives unacceptable EM structure. For the dual
LM/EM role osmium tetroxide should be avoided due to its effect on many LM stains but 1% phosphotungstic
acid (w/v) in the first absolute ethanol step of dehydration improves electron contrast without
adversely affecting most LM stains. If this does not provide adequate electron density, then “staining“ of
ultrathin sections can be carried out with osmium ( a brief exposure to 1% aqueous osmium tetroxide (Ladd
55090-55092) or osmium tetroxide (Fluka 20280) vapour on a copper grid or lead citrate.
3.3 Dehydration
A graded ethanol series is the method of choice when using LR White. Acetone acts as a radical scavenger in
the resin system and traces of acetone left in the tissue during curing can interfere with polymerisation.
3.4 Infiltration
The extreme low viscosity of LR White allows the use of short infiltration times, but these will obviously depend
on the size of the tissue. Infiltrated tissue will become translucent and sink to the bottom of the container.
A typical dehydration and infiltration schedule for a block (12x10x3mm) on a shaker would be:
•
•
•
Two changes 70% alcohol,
Two changes absolute alcohol,
Infiltrate with LR White at RT, 2-3 changes
30 mins. each.
30 mins. each.
60 mins. each or leave overnight.
3.5 Prepolymerisation
The LR White catalyst is a form of benzoyl peroxide in a solid solution to render it safe for transport. One 500 ml
bottle of LR White resin requires 9,9 g of catalyst to be added. The catalyst should be added to the resin at
room temperature and the resin must be shaken thoroughly immediately after addition of catalyst. The catalyst
will take a full 24 hours at room temperature to dissolve completely and during this time it is most helpful if the
bottle can be shaken from time to time. Do not attempt to heat the resin in order to speed the dissolution of the
catalyst.
Once mixed and fully dissolved the shelf life of the catalyzed resin stored at 40C is at least 12 months.
3.6 Polymerisation
Either heat or cold curing can be used for LM, cold curing gives slightly better cutting and staining qualities.
When cold curing it is important to cool the moulds in a bath of cold water during polymerisation, to disperse the
heat produced by the exothermic reaction, but it is not necessary to exclude oxygen from the surface of the
curing block.
Some polymerisation problems have been experienced when embedding very flat pieces of tissue which stick to
the base of the embedding mould. The way to avoid this is to smear the base of the mould with accelerator
before adding mixed resin, and allow the tissue to sink to the base of the mould rather than applying pressure.
When thermal curing, it is important to limit the contact of oxygen with resin while polymerisation occurs. The
most convenient way of achieving this is to use gelatine capsules for small pieces of tissue. Fill up to the brim
and slide the other half of the capsule on. For larger specimens, the surface of the resin must be covered and
6
one convenient method is to utilise the JB-4 type moulds, one being used as a lid for another, or to polymerise
in a nitrogen environment.
Polymerisation time and temperature are fundamental to the physical character of the final block, to a much
greater extent than with undercured epoxy systems.
We strongly recommend a temperature of 60°C ± 2°C for a period of 20-24 hours. Some ovens are not capable
of controlling temperature so closely and if faced with overbrittle blocks this is the first parameter to check.
Resin may be used straight from the refrigerator and has a very low toxicity in both monomeric and polymerised
states unlike epoxies (see Proc.Roy.Mic.Soc. 1981, 16, Pt.4, p 265-271). The cold cure accelerator does have
some toxic risk and contact with skin and eyes should be avoided.
For cold curing the accelerator should be used at one drop per 10 ml of resin and this should cause
polymerisation in 10-20 minutes. If polymerisation occurs faster than this we recommend either more careful
metering of the one drop of accelerator or a higher volume of resin per drop of accelerator.
3.7 Cutting and Mounting
Although it is possible to cut LR White on a standard microtome with a steel knife the method of choice would
be to use a heavy duty motorised microtome, and glass (Ralph type) knife.
LR White can be cut as thin as 0,25 µm on some microtomes, but it is very difficult to obtain a satisfactory stain
intensity with anything other than toluidine blue (Ladd #70975) at this thickness, simply because there is so
little tissue in the section.
For haematoxylin (Ladd #70940) and eosin (Ladd #70925) staining as well as most other routine
stains we recommend sections of 2-3 µm. It is of course possible to cut thicker (up to 15 or 20 µm) if required.
Blocks can be cut dry, the sections picked up and floated out on 30-40% acetone on a hot plate (approx. 60°
70 C), and then allow to dry at this temperature. For hard tissues, and blocks which contain a combination of
hard and soft tissues, such as marrow trephines, the following floating out fluid is recommended, again on a hot
°
plate (60-70 C).
To 20 ml acetone add 0,5 ml benzyl alhohol mix then make up to 50 ml with distilled water. A
section adhesive such as egg albumin, can be added to this if required.
3.8 Section Staining
Most routine stains give good results on tissue embedded in LR White resin using standard times and
temperatures although it may occasionally be necessary to extend some staining times e.g. methyl
green/pyronin. Stains made up in ethanol (EtOH) or methanol (MeOH) should be avoided as
these solvents soften the resin and may remove sections from the slide. Dehydration of solution through graded
alcohols after staining should also be avoided. Sections should be blotted, air dried and then mounted in a
resinous mounting medium.
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4. LR White for Immunohistochemistry
4.1 Introduction
Sections from LR White embedded tissue have been used successfully for immunocytochemistry at both the
light microscope and electron microscope levels. This demonstrates quite clearly that the visualization steps of
the immunocytochemical procedure will penetrate the resin and react with tissue antigens if they have been
preserved in the tissue.
As with all immuno-localisations the key factor is whether or not the tissue antigen has survived fixation,
processing and embedding in such a form as to be recognisable to the specific antibody. This is difficult to
predict with certainty, but some antigens have been shown to be highly resistant whilst others are fickle even in
unfixed frozen sections.
Much interest has centred on using immunocytochemistry to detect protein hormones and the various classes of
immunoglobulins and generally these classes of antigens have proved resistant to alteration both in processing
to paraffin-wax and to LR White resin.
It is the special hydrophilic nature of LR White which allows immunochemicals to permeate the suppporting
resin and reach its sites of binding and no resin pretreatment is necessary, or indeed possible, to facilitate this
penetration.
We have been successful with LR White blocks only when they have been thermally cured, probably because
when accelerator-curing the resin, the heat produced is sufficient to damage the integrity of the tissue antigen.
0
Some workers have also reported that a slight under-cure of LR White, say at 55 C for 20-24 hours aids
subsequent penetration of antisera, but we have obtained good results without deviating from the standard
polymerisation method.
If the particular antigen under consideration has already been localised in paraffin-wax sections then a trusted
fixation regime will be established and should be adhered to. For those approaching the problem for the first
time there is an extensive bibliography available regarding fixation for immunocytochemistry, much of it
contradictory, and a reference list is provided as some guidance. Rules of thumb seem to be to avoid
glutaraldehyde and perhaps use an acid rather than a neutral fixative, but there are many conflicting and
strongly held views on these topics.
Similarly, the need to enzyme digest sections prior to reaction is fraught with controversy and may indeed be
linked to the fixation regime chosen. We have used both protease type VII and trypsin type II
to good effect on our neutral buffered formalin fixed material.
If frozen, dewaxed or etched epoxy resin sections are used for immunostaining, the tissue is not surrounded by
a supporting matrix when they are being reacted. When using LR White sections the resin is still intact and
therefore diffusion to the sites of reaction must occur prior to reaction of antisera with antigen. For this reason
we have found it necessary to use antisera at approximately ten times the concentration that would work on
dewaxed sections. The exact titre of each antibody will depend upon its source and how well it has been stored,
but we have used many commercial anti-immunoglobulins at about a 1 in 10 dilution.
For the same reason the antibody stages of the reaction often benefit from a longer incubation time. Up to 2
0
hours at room temperature or overnight at 4 C in a moist chamber may be used.
Various immunoperoxidase techniques have given results on LR White tissue sections including the peroxidaseantiperoxidase complex method (PAP)(Sternberger, 1970), the hapten sandwich technique (Jasani et al., 1981),
and the indirect peroxidase method. The avidin-biotin-peroxidase complex method of Hsu has not been
successful in our hands with LR White embedded material, probably due to the molecular size of avidin. As
fairly strong antibody concentrations are required, a highly sensitive method of detection is to be prefered and
for this reason the PAP or hapten sandwich techniques are more suitable than a two layer indirect peroxidase
reaction.
Visualisation of the bound peroxidase is achieved with the diaminobenzidine-peroxidase reaction as described
by Graham and Karnovsky (1966)
Any technique where the sections are subjected to hydrogen peroxide solutions twice during staining is likely to
tend to lift sections from the slides. We have found that poly-L-lysine (MW 350'
000), is an
excellent adhesive for immunocytochemical work, and also care should be taken to dry sections onto slides very
0
thoroughly in an oven rather than a hot-plate at 60 C for two hours. This step should not have any effects on the
0
antigencity of the tissue as it will already have spent 20-24 hours at 60 C during polymerisation.
It is clear that no “standard“ immunohistochemical staining regime can be cited as there are so many variables,
but a “typical“ regime is described below for general guidance
8
4.2 PAP Procedure for LR White Sections (3µ
µm)
• Block endogenous peroxidase with 1% phenylhydrazine hydrochloride
in P.B.S (optional) 30 minutes.
• Wash in P.B.S. 1 x 5 minutes, and 2 x 5 minutes at 370C.
• 0,1% trypsin in 0,1% aqueous CaCl2
20 minutes
• Wash in ice cold distilled water.
10 minutes
• 2% Goat serum in P.B.S.
20 minutes
0
0
• First antibody (approx. 1:10 dilution) 2 hours at 37 C or overnight at 4 C
• Wash in P.B.S.
10 minutes
• Goat anti-rabbit antibody (approx. 1:20 dilution) 3 hours
0
0
at 37 C or overnight at 4 C.
• Wash in P.B.S.
10 minutes
0
0
• PAP (Sigma P 1901or P 2026) at 1:200 dilution 2 hours at 37 C or overnight at 4 C.
• Wash in P.B.S.
10 minutes.
• Wash in TRIS HCl pH 7,6
10 minutes.
• DAB/H2O
15 minutes.
• Wash in distilled water
10 minutes.
• Counterstain as required.
4.1 General Staining Procedure – Indirect Technique
• Block endogeneous peroxidase with 0,3% H2O2 in 50% methanol
• Block only when peroxidase conjugated secondary antibodies are used.
• Block with levamisole when alkaline
phosphatase conjugated secondary antibodies are used.
• Rinse in P.B.S.
2x
• Treat sections according to the antigen retrieval technique indicated below.
• Rinse in P.B.S.
2x
• Incubate with primary antibody, suitably diluted in P.B.S.
(see product information sheet). Optimal dilution should be tested in
combination with second antibody.
• Rinse in P.B.S.
2x
• Incubate with conjugated second antibody.
• Dilute second antibody suitably with P.B.S. Second antibody must be
directed against antibodies of the species in which the primary antibody
was raised.
• After incubation with fluorochrome labeled antibodies, sections are rinsed
in P.B.S. and mounted in a mounting media such as DPX,
Gelvatec, Gelvastab, PVA-DAPCO or PVA-NDP.
• After incubation with enzyme conjugated antibodies (peroxidase,
alkaline phosphatase) rinse in P.B.S.
• Apply appropriate chromogen substrate.
• Rinse and counterstain, mount sections.
20 minutes
5 minutes
5 minutes
5 minutes
30 minutes
4.2 Antigen Retrievals
• Proteolytic enzyme treatment
• Trypsin solution: 0,1% trypsin + 0,1% CaCl2 Dissolve in distilled water, adjust to pH 7,8.
• Pepsin solution: 0,4% pepsin. Dissolve in 0,01N HCl. Incubation time in pepsin solution up to
2 hours at RT.
• Pronase solution: 0,1% pronase + 0,01% CaCl2 Incubation time in pronase solution 10
0
minutes at 37 C
• Microwave oven treatment
• Place slides in a coplin jar containing sodium citrate buffer
pH 6,0. Incubate for 3 x 5 minutes in a microwave oven, 750 W, with 1 minutes intervals.
• Rinse for 30 minutes in P.B.S. at RT.
For immunocytochemistry LR White must be thermally cured and not accelerator cured!
9
5. LR White for Hard Tissue
5.1 Introduction
LR White can be used for the microtomy of decalcified bone and teeth and also for microtomy or “sawing and
grinding” of non-decalcified tissues.
5.2 Decalcified Tissue
May be processed, cut and stained similarly to soft tissue (see LR White for Light Microscopy), except that
dehydration and infiltration times may need to be extended depending on the size of tissue. It is also
recommended that bone be “de-fatted” to improve the penetration of resin into marrow cavities.. This can be
achieved by using chloroform (Fluka 25691) after dehydration, returning to absolute alcohol to remove the
chloroform before infiltration with resin and polymerising.
5.3 Non-decalcified Tissue
Dehydration and infiltration times will vary depending on size and density of tissue. Those laboratories using
methyl- or butylmethacrylate (Ladd #21335 or 21330 respectively) at present can use similar dehydration
times, but infiltration will probably be shortened due to the low viscosity of the resin.
5.4 Dehydration
A graded series of alcohols should be used for dehydration of tissue, and when processing bone “de-fatting” is
recommended to improve the penetration of resin into the marrow cavities. This can be done using chloroform
for the same length of time that would be necessary to clear the tissue. The bone should then be
taken back to absolute alcohol and given sufficient changes to remove the chloroform before infiltration with LR
White.
5.5 Infiltration
Several changes of resin will be necessary and impregnation under vacuum is recommended.
5.6 Prepolymerisation
The LR White catalyst is a form of benzoyl peroxide in a solid solution to render it safe for transport. One 500 ml
bottle of LR White resin requires 9,9 g of catalyst to be added. The catalyst should be added to the resin at
room temperature and the resin must be shaken thoroughly immediately after addition of catalyst. The catalyst
will take a full 24 hours at room temperature to dissolve completely and during this time it is most helpful if the
bottle can be shaken from time to time. Do not attempt to heat the resin in order to speed the dissolution of the
catalyst.
°
Once mixed and fully dissolved the shelf life of the catalyzed resin stored at 4 C is at least 12 months.
5.7 Polymerisation
The tissue can be heat or accelerator cured after embedding in strong plastic moulds, such as the JB-4 or Peela-way type, or in aluminium foil dishes (Ladd #20620).
When heat curing, the moulds should first be filled with resin then the tissue added and orientated.
0
0
Polymerisation will occur in 18-24 hours at 60 C ± 2 C. The surface of the block exposed to oxygen may remain
slightly sticky, but this will not affect the cutting quality of the face of the block. Some ovens are not capable of
controlling temperature so closely and if faced with overbrittle blocks this is the first parameter to check.
When accelerator or “cold” curing, the moulds should be placed in a bath of ice-cold water to disperse the heat
produced during the exothermic polymerisation. The base of the moulds should first be smeared with
accelerator using a cotton-wool bud or swab, the accelerator is then added to the resin, one drop per 10 ml
resin, and thoroughly mixed before pouring into the mould, the tissue is then placed into the mould and
orientated. Polymerisation should occur in 10-20 minutes, if its occurs faster than this we recommend either
more careful metering of the one drop of accelerator or a higher volume of resin per drop of accelerator.. N.B.
The accelerator is toxic and contact with skin and eyes should be avoided.
5.8 Cutting and Mounting
Bone marrow trephines and small pieces of cancellous bone, cortical bone and teeth offer too much resistance
to the microtome knife and preparations of this material must be prepared by sawing and grinding.
5.9 Microtomy
Sections can be cut, using Ralph type glass knifes for trephines or a tungsten carbide knife for larger pieces of
cancellous bone, from 2-10 µm. Blocks can be cut dry, the sections picked up and floated out on a hot plate at
0
60-70 C using the following solution:
10
20 ml acetone (Ladd #20415) add 0,5 ml benzyl alcohol mix then make up to 50 ml with distilled
water. A section adhesive such as egg albumin can be added to this if required. Sections should be allowed to
dry on a hot plate for at least 30 minutes before staining.
5.10 Sawing and Grinding
Thick slices 150-200 µm can be cut using a milling machine and then ground to the required thickness, usually
20 µm for staining or 70 µm for microradiography; the section is inclined to fragment if grinding is continued
much below 20 µm.
Using the newer types of saw microtome, such as the Leitz 1600 which has a diamond-coated-internal-holesaw, sections can actually be cut at 20 µm and no further grinding is necessary.
5.11 Section Staining
Sections of material embedded in LR White are stained “free floating”, times of staining are usually longer than
those for paraffin sections, and dehydration through alcohols should be avoided. A recommended method for
haematoxylin and eosin staining is as follows:
•
Remove calcium deposits, which would otherwise interfere with the staining, from the sections by treating
TM
rapid decalcifying solution
with Kristensen’s decalcifying solution or Accumate RDO
for about 15 minutes.
•
Wash in running tap water for a few minutes to remove the formic acid from the tissue.
•
Transfer the section to several changes of distilled water, a few minutes each and then into a 0,5% w/v
solution of periodic acid in distilled water where it should be left for 5 minutes.
•
Wash the section with several changes of distilled water and stain with Harris haematoxylin
for about 1 hour.
•
Transfer the section, after a short time in distilled water, into running tap water to “blue” the haematoxylin
stained tissue.
•
Check the haematoxylin stain; if the tissue should be overstained or the surface of the resin has become
stained with haematoxylin, this may be corrected by a short differentiation in acid alcohol (0,5% HCl in 70%
alcohol) and “reblueing” of the stained tissue.
•
Rinse the section in distilled water and counterstain, using a 5% solution of eosin Y in distilled
water; leave the section in the eosin stain for 30 minutes to 1 hour, wash briefly in running tap water and
check the staining of the tissue. Nuclei and haematoxophilic elements should be bright blue, cytoplastic
structures in various shades of red-pink.
•
Rinse the section in distilled water, blot dry with filter paper and either clear briefly in xylene
or xylene alternative and mount in a mounting media such as DPX, Gelvatec,
Gelvastab, PVA-DAPCO or PVA-NPD, or directly in LR White resin by adding a drop of accelerator to 1 ml
of resin.
III. Ordering information
Cat.No.: 12390
Cat.No.: 12392
Cat.No.: 12393
Cat.No.: 12394
Cat.No.: 12398
Comes With Resin
LR White Embedding Kit, Hard Grade
LR White Embedding Kit , Medium Grade
LR White Resin, Hard Grade
LR White Resin, Medium Grade
LR White Accelerator
LR White Catalyst
11
500 ml
500 ml
10 ml
10 g
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