Epithelial Antigen Antibody Staining Protocol for Immunohistochemistry


Description: Monoclonal Anti-Epithelial Antigen, Clone Ber-EP4 (Anti-epithelial antigen) reacts with the peptide portion of a 34 kDa and 39 kDa glycoprotein expressed on the membrane and in the cytoplasm of normal epithelial cells and carcinomas of various origins. In immunoprecipitation studies, antiepithelial antigen has been shown to precipitate a 34 kDa and a 39 kDa polypeptide from radiolabelled MCF-7 cells. Under non-reducing conditions the molecular weight was found to be slightly higher (39 kDa and 44 kDa). Deglycosylation of the precipitate reduced the molecular weight to 31 kDa and 36 kDa respectively. Internal labelling of MCF-7 and K562 cells for up to 24 hours resulted in the precipitation of only one chain (39 kDa) after two hours of labelling, suggesting a 39 kDa gene product for both chains. Anti-epithelial antigen, Clone Ber-EP4 has been shown to be equivalent in specificity to monoclonal antibody HEA15.1-3 Sequential immunoprecipitation and cross-blocking experiments demonstrated that both antibodies recognize the same antigen.


Primary Antibody

Name: Epithelial Antigen Antibody

Clone:  Ber EP4, Mouse anti-Human

Supplier: Dako

Catalog Number: M0804

Dilution: 1:20 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) or IHC-TekTM Proteinase K Solution (Cat# IW-1101)
Heat/Cool Temperature: 95 ºC - 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: Membrane/cytoplasmic
Images: Search image

Additional Information:
Tissue Type: Pancreas, pituitary gland, small intestine, colon, kidney.
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Pancreas, pituitary gland, small intestine, colon, kidney.
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


No need to perform antigen retrieval on frozen sections