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

Name: Cytokeratin Antibody

Clone: AE1/AE3, Mouse anti-Human

Supplier: Dako

Catalog Number: M3515

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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


There is no need to perform antigen retrieval for frozen sections.