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

Name: p53 (DO-1) Antibody

Clone: Mouse monoclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-126

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.

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: