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eNOS Antibody Staining Protocol for Immunohistochemistry
Description: Endothelial nitric-oxide synthase (eNOS) is an important enzyme in the cardiovascular system. It catalyzes the production of nitric oxide (NO), a key regulator of blood pressure, vascular remodeling and angiogenesis. eNOS is regulated by phosphorylation at multiple sites. The two most throughly studied sites are the activation site serine 1177 and the inhibitory site threonine 495. Several protein kinases including Akt/PKB, PKA and AMPK activate eNOS by phosphorylating Ser1177 in response to various stimuli. In contrast, bradykinin and hydrogen peroxide activate eNOS activity by promoting Thr495 dephosphorylation.
Primary Antibody
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Name:
Phospho-eNOS (Ser1177) |
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Clone: Rabbit polyclonal |
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Supplier: Cell Signaling Technology |
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Catalog Number: 9571L |
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Dilution: 1:10 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 minutes/room temperature |
Antigen Retrieval
| Device: Antigen retrieval is not needed |
| Buffer/pH value: N/A |
| Heat/Cool Temperature: N/A |
| Heat/Cool Time: N/A |
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: Cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Heart |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Heart |
| Negative Control: Omit primary antibody, isotype control or 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 |
Notes:
Not tested on frozen sections |
References: