p21 Antibody Staining Protocol for Immunohistochemistry


Description: It is now well established that cyclins play a positive role in promoting cell cycle transitions via their ability to associate with and activate their cognate cyclin-dependent kinases (Cdks). Cdk2 associates with cyclins A, D and E and has been implicated in the control of the G1 to S phase transition in mammals. A novel 21 kDa Cdk-interacting protein, designated Cip1, has been identified in cyclin A, cyclin D1, cyclin E and Cdk2 immunoprecipitates. p21 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4 and cyclin D2-Cdk4 complexes. Expression of p21 (also designated WAF1/Cip1) is inducible by wild type, but not mutant, p53. The mouse homolog of p21 has been identified as a 20 kDa protein designated CAP20.


Primary Antibody

Name: p21 (F-5) Antibody

Clone: Mouse anti-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-6246

Dilution: 1:10 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
Fixation: Formalin fixed paraffin sections
Positive Control: Pancreas, liver, lung, breast
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