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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
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Name: NFkB p65 (F-6) Antibody |
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Clone: F-6, Mouse anti-Human |
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Supplier: Santa Cruz Biotechnology |
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Catalog Number: sc-8008 |
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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. |
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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: 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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
References:
1. Sung-Hsin Kuo, et al. (2004) Nuclear Expression of BCL10 or Nuclear Factor Kappa B Predicts Helicobacter pylori–Independent Status of Early-Stage, High-Grade Gastric Mucosa-Associated Lymphoid Tissue Lymphomas. Journal of Clinical Oncology, Vol 22, No 17 (September 1), 2004: pp. 3491-3497
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