p53 Antibody Staining Protocol for Immunohistochemistry


Description: Mutation of the p53 protein may represent the commonest genetic event in human malignancy. Mutation results in the overexpression and thus detectability of the protein by immunohistochemistry. p53 appears to function both as an oncoprotein, perhaps by complexing in its mutant form with wild type p53, and as a tumour suppressor gene in its normal state.


Primary Antibody

Name: p53 Antibody

Clone: Rabbit anti-Human

Supplier: Novocastra

Catalog Number: NCL-p53-CM1

Dilution: 1:50 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 min/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: Nuclear
Images: Search image

Additional Information:
Species Reactivity: Human, mouse
Fixation: Formalin fixed paraffin sections, acetone fixed frozen sections
Positive Control: Breast or colonic carcinomas
Negative Control: Omit primary antibody, isotype control, 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