NFkB p65 Antibody Staining Protocol for Immunohistochemistry


Description: Proteins encoded by the v-Rel viral oncogene and its cellular homolog, c-Rel, are members of a family of transcription factors that include the two subunits of the transcription factor NFkB (p50 and p65) and the Drosophila maternal morphogen, dorsal. Both proteins specifically bind to DNA sequences that are the same or slight variations of the 10 bp kB sequence in the immunoglobulin ê light chain enhancer. This same sequence is also present in a number of other cellular and viral enhancers. The DNA-binding activity of NFkB is activated and rapidly transported from the cytoplasm to the nucleus in cells exposed to mitogens or growth factors. cDNAs encoding precursors for two distinct 50 kDa proteins have been described, designated p105 and p100, respectively. The p105 precursor contains p50 at its amino terminus and a C-terminal region that when expressed as a separate molecule, designated pdI, binds to p50 and regulates its activity.


Primary Antibody

Name: NFkB p65 (C-20) Antibody

Clone: C-20, Rabbit anti-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-372

Dilution: 1:50 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/cytoplasmic
Images: Search image

Additional Information:
Species Reactivity: Human, rat, mouse
Fixation: Formalin fixed paraffin sections
Positive Control: Human Gastric cancer, cervical cancer, prostate 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