Cytokeration (High Molecular Weight) Antibody Staining Protocol for Immunohistochemistry


Description: Cytokeratins are intermediate filament cytoskeletal proteins essential to development and differentiation of epithelial cells. Approximately twenty different cytokeratins have been identified and are classified and numbered according to molecular weight and isoelectric points. In general, most low molecular weight cytokeratins (40-54 kDa) are distributed in nonsquamous epithelium, Monoclonal mouse anti-human cytokeratin, High Molecular Weight (HMW), clone 34BE12 (anti-CK, HMW, 34BE12) is to identify the 66, 57, 51 and 49 kDa1 proteins corresponding to cytokeratins 1, 5, 10 and 14 of the Moll catalog, in normal and neoplastic tissues in frozen or formalin-fixed paraffin-embedded tissue using immunohistochemical methods.


Primary Antibody

Name: Cytokeratin (High Molecular Weight) Antibody

Clone: 34BE12, Mouse anti-Human

Supplier: Dako

Catalog Number: M0630

Dilution: 1:500 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: Epithelium in skin, breast, lung, kidney, pancreas, thymus, tonsil.
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections.
Positive Control: Epithelium in skin, breast, lung, kidney, pancreas, thymus, 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.