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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
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Name: p21 (F-5) Antibody |
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Clone: Mouse anti-Human |
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Supplier: Santa Cruz Biotechnology |
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Catalog Number: sc-6246 |
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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. |
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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 |
Counterstain
| Reagent: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
References: