NOS3 Antibody Staining Protocol for Immunohistochemistry


Description: Nitric oxide (NO) has a broad range of biological activities and has been implicated in signaling pathways in phylogenetically diverse species. Nitric oxide synthases (NOSs), the enzymes responsible for synthesis of NO, contain an N-terminal oxygenase domain and a C-terminal reductase domain. NOS activity requires homodimerization as well as three cosubstrates (L-arginine, NADPH and O2) and five cofactors or prosthetic groups (FAD, FMN, calmodulin, tetrahydrobiopterin and heme). Several distinct NOS isoforms have been described and been shown to represent the products of three distinct genes. These include two constitutive Ca++/CaM-dependent forms of NOS, including ncNOS (also designated NOS1) whose activity was first identified in neurons and maps at 12q24.2, and ecNOS (also designated NOS3), first identified in endothelial cells and mapping at 7q35-36. The inducible form of NOS, iNOS (also designated NOS2), is Ca++-independent, expressed in a broad range of cell types and maps to 17cen -q12.


Primary Antibody

Name: NOS3 Antibody

Clone: Rabbit polyclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-654

Dilution: 1:500 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: 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 (endothelial cell)
Images: Search image

Additional Information:
Species Reactivity: Human, rat, mouse
Fixation: Formalin fixed paraffin sections
Positive Control: Kidney, brain
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