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Cytokeration Antibody Staining Protocol for Immunohistochemistry
Description: The cytokeratins are a family of water-soluble proteins with molecular weights between 40-70 kDa that form the cytoskeleton of epithelial cells. At least 19 different cytokeratins have been identified and can be divided into two subfamilies. Subfamily A comprises relatively acidic cytokeratins (with a pI under 5.5) whereas members of subfamily B have a relatively basic pI of 6 or over. Monoclonal mouse anti-human Cytokeratin, Clones AE1/AE3 and is to identify two epitopes present on a majority of epithelial cytokeratins in acetone-fixed frozen and formalin-fixed, paraffin-embedded tissue using immunohistochemical methods.
Primary Antibody
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Name: Cytokeratin Antibody |
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Clone: AE1/AE3, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M3515 |
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Dilution: 1:100 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60 minutes/room temperature |
Antigen Retrieval
| Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) |
| Heat/Cool Temperature: 95-100 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Cervix, colon, esophagus, skin, small intestine, stomach, and tonsil. |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections. |
| Positive Control: Cervix, colon, esophagus, skin, small intestine, stomach, and tonsil. |
| Negative Control: Omit primary antibody, isotype control or absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
Notes:
There is no need to perform antigen retrieval for frozen sections. |
References: