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

Name: Phospho-eNOS (Ser1177) Antibody

Clone: Rabbit polyclonal

Supplier: Cell Signaling Technology

Catalog Number: 9571L

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.

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: