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p53 Antibody Staining Protocol for Immunohistochemistry
Description:
p53, a DNA-binding, oligomerization domain and
transcription activation domain-containing tumor suppressor,
upregulates growth arrest and apoptosisrelated 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 MDM-2, an E3 ubiquitin ligase that is upregulated in the
presence of active p53, where MDM-2 poly-ubiquitinates p53 for
proteasome targeting. p53 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 (DO-1) Antibody |
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Clone: Mouse monoclonal |
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Supplier: Santa Cruz Biotechnology |
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Catalog Number: sc-126 |
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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 |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Breast cancer |
| 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: