Nitrotyrosine Antibody Staining Protocol for Immunohistochemistry


Description: Nitric oxide (NO) is a product of the enzymatic conversion of arginine to citrulline by nitric oxide synthase. NO reacts rapidly with superoxide (6.7 x 109 M-1sec-1) to form peroxynitrite. At physiological pH and in the presence of transition metals, peroxynitrite undergoes heterolytic cleavage to form hydroxyl anion and nitronium ion, the latter of which nitrates protein tyrosine residues. Thus, the presence of nitrotyrosine on proteins can be used as a marker for peroxynitrite formation in vivo. Nitrotyrosine has been shown to be present in proteins from a variety of clinical conditions including atherosclerotic lesions of human coronary arteries, postischemic heart, and placenta during preeclampsia. Increased nitration of proteins in motor neurons has been identified in patients with ALS (amyotrophic lateral sclerosis) and may be due to mutations in superoxide dismutase.


Primary Antibody

Name: Nitrotyrosine Antibody

Clone: Rabbit polyclonal

Supplier: Upstate

Catalog Number: 06-284

Dilution: 1:200 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
Images: Search image

Additional Information:
Species Reactivity: Human, mouse, rat
Fixation: Formalin fixed paraffin sections
Positive Control: Acutely injured human lung, ischemic 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