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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
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Name: Phospho-NF-kB p65 Antibody |
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Clone: Rabbit anti-Human |
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Supplier: Cell Signaling Technology |
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Catalog Number: 3031 |
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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. |
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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, 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 |
References: