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p53 Antibody Staining Protocol for Immunohistochemistry
Description: p53 is a DNA-binding, oligomerization domain- and transcription activation domain-containing tumor suppressor that upregulates growth arrest and apoptosis-related genes in response to stress signals, thereby influencing programmed cell death, cell differentiation, and cell cycle control mechanisms. p53 localizes to the nucleus yet can be chaperoned to the cytoplasm by the negative regulator MDM2, an E3 ubiquitin ligase that is upregulated in the presence of active p53, where MDM2 polyubiquitinates p53 for proteasome targeting. p53 can assemble into tetramers in the absence of DNA, fluctuates between latent and active (DNA-binding) conformations, and is differentially activated through post- translational modifications including phosphorylation and acetylation. Mutations in the DNA-binding domain (DBD) (amino acids 110-286) of p53 can compromise energetically favorable association with cis elements and are implicated in several human cancers.
Primary Antibody
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Name: p53 (FL-393) Antibody |
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Clone: Rabbit Polyclonal |
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Supplier: Santa Cruz Biotechnology |
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Catalog Number: sc-6243 |
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Dilution: 1:100 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: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Nuclear |
| Images: Search image |
Additional Information:
| Species Reactivity: Human, mouse, rat |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Human breast carcinoma, mouse skin tumor. |
| 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: