Phospho-NF-kB p65 Antibody Staining Protocol for Immunohistochemistry


Description: Transcription factors of the nuclear factor kB (NF-kB)/Rel family play a pivotal role in inflammatory and immune responses. There are five family members in mammals; RelA, c-Rel, RelB, NF-kB1 (p105/p50), and NF-kB2 (p100/p52). p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. The p50 and p52 products form dimeric complexes with Rel proteins, which  are then able to bind DNA and regulate transcription. In unstimulated cells, NF-kB is sequestered in the cytoplasm by its inhibitory proteins, the IkBs. NF-kBactivating agents can induce the phosphorylation of IkBs, which targets them for rapid degradation through a ubiquitin-proteasome pathway, releasing NF-kB to enter the nucleus and regulate gene expression. Processing of NF-kB2 is regulated by IKK1 (IKK-a), which triggers the phosphorylation and processing to p52, which can then undergo nuclear translocation.


Primary Antibody

Name: Phospho-NF-kB p65 Antibody

Clone: Rabbit anti-Human

Supplier: Cell Signaling Technology

Catalog Number: 3031

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: 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, mouse
Fixation: Formalin fixed paraffin sections
Positive Control: Breast carcinoma
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