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Cytokeration 8&18 Antibody Staining Protocol for Immunohistochemistry
Description: Cytokeratins are a family of water-insoluble intermediate filaments, composed of polypeptide chains found in the cytoskeleton of epithelial cells. Twenty different forms of human cytokeratins exist (Moll numbers 1-20) in both normal and malignant epithelial cell lines. Specific combinations of cytokeratin peptides are associated with certain epithelial cells, and are therefore useful in the characterization of poorly differentiated carcinoma. Cytokeratins may also be clinically significant indicators in distinguishing carcinoma from malignant tumors of non-epithelial origin, such as lymphoma, sarcoma, and melanoma.
Primary Antibody
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Name: Cytokeratin 8&18 Antibody |
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Clone: 5D3, Mouse anti-Human |
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Supplier: Novocastra Labs |
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Catalog Number: NCL-5D3 |
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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: Incubator |
| Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101) |
| Heat/Cool Temperature: 37 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: Skin |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections. |
| Positive Control: Skin |
| 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: