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 Drosophilamaternal 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 k 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 (F-6) Antibody

Clone: F-6, Mouse anti-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-8008

Dilution: 1:200 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: 30-60 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



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2. Ross JS, et al. (2004) Expression of nuclear factor-kappa B and I kappa B alpha proteins in prostatic adenocarcinomas: correlation of nuclear factor-kappa B immunoreactivity with disease recurrence. Clin Cancer Res 2004 Apr 1;10(7):2466-72

3. Nair A, et al. (2003) NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix. Oncogene. 2003 Jan 9;22(1):50-8

4. Lee BL, et al. (2005) Nuclear factor-kappaB activation correlates with better prognosis and Akt activation in human gastric cancer. Clin Cancer Res. 2005 Apr 1;11(7):2518-25

5. Zhou, JY, et al (2004) Effect of a-pinene on nuclear translocation of NF-kB in THP-1 cells. Acta Pharmacol Sin 2004 Apr; 25 (4): 480-484