p21 Antibody Staining Protocol for Immunohistochemistry


Description: p21WAF1/Cip1/Sdi1/Pic1 is a tumor suppressor protein. Expression of p21WAF1 is induced by wild type, but not mutant, p53 suppressor protein. The p21WAF1 protein binds to cyclin/CDK complexes and inhibits their kinase activity thereby stopping cell cycle progression. Meanwhile, the p21WAF1 protein also binds to PCNA (proliferating cell nuclear antigen) and blocks DNA replication but not the DNA repair process. Evidently, inactivation of p53 disrupts the p53-dependent expression of p21WAF1. p21WAF1is a tumor suppressor involved in the pathogenesis of a variety of malignancies.


Primary Antibody

Name: p21 (WAF1) Antibody

Clone: Rabbit anti-Mouse

Supplier: Lab Vision

Catalog Number: RB-032-P

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: Rat, mouse
Fixation: Formalin fixed paraffin sections
Positive Control: Colon, NIH3T3 or MAD109 cells.
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