eNOS Antibody Staining Protocol for Immunohistochemistry


Description: Nitric oxide synthase NOS oxidizes a guanidine nitrogen of arginine releasing nitric oxide in the form of a free radical and citrulline. Nitric oxide thus generated acts as a messenger in diverse functions including vasodilation neurotransmission, anti tumor and anti pathogenic activities. NOS is classified under three types: neuronal NOS (nNOS) or brain NOS (bNOS); inducible NOS (iNOS) or macrophage NOS (mNOS); and endothelial NOS (eNOS).


eNOS is a calcium/calmodulin dependent enzyme which undergoes several post translational modifications, including acylation with myristate and palmitate, and phosphorylation on numerous residues. As with the other members of the NOS family, eNOS derives the diffusible multifunctional second messenger NO from L arginine through a series of reactions in which L citrulline is an intermediate. eNOS plays an important role in controlling vascular tone, platelet aggregation, and cardiac myocyte function.


Primary Antibody

Name: eNOS Antibody, prediluted

Clone: Rabbit polyclonal

Supplier: Abcam

Catalog Number: ab15280

Dilution: prediluted ready to use

Incubation Time/Temp: 60 minutes/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 ºC/Room Temperature
Heat/Cool Time: 20 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: Cytoplasmic of endothelial cells
Images: Search image

Additional Information:
Species reactivity: Human, mouse, rat, cow, dog and pig
Fixation: Formalin fixed paraffin sections
Positive Control: HUVEC cells, placenta, skin
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


Not tested on frozen sections