Data Sheet

Data Sheet
Pan Cytokeratin Plus [AE1/AE3+8/18]
Concentrated and Prediluted Cocktail Antibody
Control Number: 901-162-032415
Catalog Number:
CM 162 A, B, C
PM 162 AA, H
IP 162 G10
OAI 162 T60
Description:
0.1, 0.5, 1.0 ml, concentrated
6.0, 25 ml, prediluted
10 ml, prediluted
60 tests, prediluted
Dilution:
1:50-1:100
Ready-to-use
Ready-to-use
Ready-to-use
Diluent:
Da Vinci Green
N/A
N/A
N/A
Intended Use:
For In Vitro Diagnostic Use
Pan Cytokeratin Plus [AE1/AE3+8/18] is a mouse monoclonal antibody cocktail that is
intended for laboratory use in the qualitative identification of a broad spectrum of
acidic and basic cytokeratin proteins by immunohistochemistry (IHC) in formalin-fixed
paraffin-embedded (FFPE) human tissues. The clinical interpretation of any staining or
its absence should be complemented by morphological studies using proper controls
and should be evaluated within the context of the patient’s clinical history and other
diagnostic tests by a qualified pathologist.
Summary and Explanation:
AE1/AE3 recognizes acidic and basic subfamilies of cytokeratins. The cocktail of
these two antibodies can be used to detect most human epithelia. The acidic
cytokeratins have molecular weights of 56.5, 55, 51, 50, 50, 48 46, 45, and 40 kDa.
The basic cytokeratins have molecular weights of 65-67, 64, 59, 58, 56 and 52 kDa.
Clone 5D3 recognizes cytokeratin (CK) 8 and 18 intermediate filament proteins.
These are 52.5 kDa and 45 kDa respectively. In normal tissues, 5D3 recognizes all
simple and glandular epithelium. In the past, AE1/AE3 has had problems marking
certain tissues types and adenocarcinomas. The addition of CK 8/18 remedies some of
these problems. For example, a study of twenty-eight lipid cell (steroid cell) tumors of
the ovary were studied by immunohistochemistry; 46% were positive for Cytokeratin
8/18 antibody, 37% were positive with the Cytokeratin cocktail AE1/AE3.
Principle of Procedure:
Antigen detection in tissues and cells is a multi-step immunohistochemical process.
The initial step binds the primary antibody to its specific epitope. A secondary antibody
may be applied to bind the primary antibody, followed by an enzyme labeled polymer;
or an enzyme labeled polymer may be applied directly to bind the primary antibody.
The detection of the bound primary antibody is evidenced by an enzyme-mediated
colorimetric reaction.
Source: Mouse monoclonal
Species Reactivity: Human, mouse and rat
Clone: AE1/AE3 + 5D3
Isotype: IgG1
Total Protein Concentration: ~10 mg/ml. Call for lot specific Ig concentration.
Epitope/Antigen: AE1/AE3 + CK8/18
Cellular Localization: Cytoplasmic
Positive Control: Skin or adenocarcinoma
Known Applications:
Immunohistochemistry (formalin-fixed paraffin-embedded tissues)
Supplied As: Buffer with protein carrier and preservative
Storage and Stability:
Store at 2ºC to 8ºC. Do not use after expiration date printed on vial. If reagents are
stored under conditions other than those specified in the package insert, they must be
verified by the user. Diluted reagents should be used promptly; any remaining reagent
should be stored at 2ºC to 8ºC.
Protocol Recommendations (intelliPATH and manual use):
Peroxide Block: Block for 5 minutes with Biocare's Peroxidazed 1.
Pretreatment Protocol:
Pretreatment may be performed by heat retrieval or enzyme digestion.
Heat Retrieval Method:
Pretreatment Solution (recommended): Reveal
Retrieve sections under pressure using Biocare's Decloaking Chamber, followed by a
wash in distilled water. Allow solution to cool for 10 minutes then wash in distilled
water.
Digestion Method:
Digest with Trypsin enzyme for 5 minutes at 37ºC or for 20 minutes at RT.
ISO
9001&13485
CERTIFIED
Protocol Recommendations (intelliPATH and manual use) Cont'd:
Protein Block (Optional): Incubate for 5-10 minutes at RT with Biocare's Background
Punisher.
Primary Antibody: Incubate for 30 minutes at RT.
Probe: Incubate for 10 minutes at RT with a secondary probe.
Polymer: Incubate for 10-20 minutes at RT with a tertiary polymer.
Chromogen:
Incubate for 5 minutes at RT with Biocare's DAB - OR - Incubate for 5-7 minutes at
RT with Biocare's Warp Red.
Counterstain:
Counterstain with hematoxylin. Rinse with deionized water. Apply Tacha's Bluing
Solution for 1 minute. Rinse with deionized water.
intelliPATH™ Automated Slide Stainer:
IP162 is intended for use on the intelliPATH™ Automated Slide Stainer. Refer to the
intelliPATH Automated Slide Stainer manual for specific instructions on its use. When
using the intelliPATH, peroxide block with intelliPATH Peroxidase Blocking Reagent
(IPB5000) may be performed following pretreatment.
Protocol Recommendations (ONCORE Automated Slide Staining System):
OAI162 is intended for use with the ONCORE Automated Slide Staining System.
Refer to the ONCORE Automated Slide Staining System User Manual for specific
instructions on its use. Protocol parameters in the ONCORE Automated Slide Stainer
Protocol Editor should be programmed as follows:
Protocol Name: Pan CK Plus
Protocol Template (Description): Ms HRP Template 1
Dewaxing (DS Option): DS Buffer
Antigen Retrieval (AR Option): AR2, low pH; 90°C
Reagent Name, Time, Temp.: Pan CK Plus, 30 min., 25°C
Technical Note:
1. With cytokeratin markers, heat retrieval may provide a higher sensitivity assay;
whereas, enzyme digestion may produce greater specificity. Users should validate the
pretreatment method for their specific application.
2. This antibody has been optimized for use with Biocare's MACH 4 Universal HRPPolymer Detection, intelliPATH Universal HRP Detection Kit and ONCORE HRP
Detection. Other Biocare polymer detection kits may be used; however, users must
validate incubation times and protocols for their specific application. Use TBS for
washing steps.
Limitations:
The optimum antibody dilution and protocols for a specific application can vary. These
include, but are not limited to fixation, heat-retrieval method, incubation times, tissue
section thickness and detection kit used. Due to the superior sensitivity of these unique
reagents, the recommended incubation times and titers listed are not applicable to other
detection systems, as results may vary. The data sheet recommendations and protocols
are based on exclusive use of Biocare products. Ultimately, it is the responsibility of
the investigator to determine optimal conditions. The clinical interpretation of any
positive or negative staining should be evaluated within the context of clinical
presentation, morphology and other histopathological criteria by a qualified
pathologist. The clinical interpretation of any positive or negative staining should be
complemented by morphological studies using proper positive and negative internal
and external controls as well as other diagnostic tests.
Quality Control:
Refer to CLSI Quality Standards for Design and Implementation of
Immunohistochemistry Assays; Approved Guideline-Second edition (I/LA28-A2).
CLSI Wayne, PA, USA (www.clsi.org). 2011.
Page 1 of 2
Pan Cytokeratin Plus [AE1/AE3+8/18]
Concentrated and Prediluted Cocktail Antibody
Control Number: 901-162-032415
Precautions:
1. This antibody contains less than 0.1% sodium azide. Concentrations less than 0.1%
are not reportable hazardous materials according to U.S. 29 CFR 1910.1200, OSHA
Hazard communication and EC Directive 91/155/EC. Sodium azide (NaN3) used as a
preservative is toxic if ingested. Sodium azide may react with lead and copper
plumbing to form highly explosive metal azides. Upon disposal, flush with large
volumes of water to prevent azide build-up in plumbing. (Center for Disease Control,
1976, National Institute of Occupational Safety and Health, 1976) (6)
2. Specimens, before and after fixation, and all materials exposed to them should be
handled as if capable of transmitting infection and disposed of with proper precautions.
Never pipette reagents by mouth and avoid contacting the skin and mucous membranes
with reagents and specimens. If reagents or specimens come into contact with sensitive
areas, wash with copious amounts of water. (7)
3. Microbial contamination of reagents may result in an increase in nonspecific
staining.
4. Incubation times or temperatures other than those specified may give erroneous
results. The user must validate any such change.
5. Do not use reagent after the expiration date printed on the vial.
6. The SDS is available upon request and is located at http://biocare.net/.
ISO
9001&13485
CERTIFIED
Troubleshooting:
Follow the antibody specific protocol recommendations according to data sheet
provided.
If atypical results occur, contact Biocare's Technical Support at
1-800-542-2002.
References:
1. Seidman JD, Abbondanzo SL, Bratthauer GL. Lipid cell (steroid cell) tumor of the
ovary: immunophenotype with analysis of potential pitfall due to endogenous biotinlike activity. Int J Gynecol Pathol. 1995 Oct; 14(4):331-8.
2. Bunton TE. The immunocytochemistry of cytokeratin in fish tissues. Vet Pathol.
1993 Sep; 30(5):418-25.
3. Sorensen SC, et al. Structural distinctions among human breast epithelial cells
revealed by the monoclonal antikeratin antibodies AEI and AE3. J Pathol. 1987 Oct;
153(2):151-62.
4. Pinkus GS, Etheridge CL, O'Connor EM. Are keratin proteins a better tumor marker
than epithelial membrane antigen? A comparative immunohistochemical study of
various paraffin-embedded neoplasms using monoclonal and polyclonal antibodies.
Am J Clin Pathol. 1986 Mar; 85(3):269-77.
5. Pinkus GS, et al. Optimal immunoreactivity of keratin proteins in formalin-fixed,
paraffin-embedded tissue requires preliminary trypsinization. An immunoperoxidase
study of various tumours using polyclonal and monoclonal antibodies. J Histochem
Cytochem. 1985 May; 33(5):465-73.
6. Center for Disease Control Manual. Guide: Safety Management, NO. CDC-22,
Atlanta, GA. April 30, 1976 "Decontamination of Laboratory Sink Drains to Remove
Azide Salts."
7. Clinical and Laboratory Standards Institute (CLSI). Protection of Laboratory
Workers from Occupationally Acquired Infections; Approved Guideline-Fourth Edition
CLSI document M29-A4 Wayne, PA 2014.
Page 2 of 2
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