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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
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Name: Nitrotyrosine Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Upstate |
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Catalog Number: 06-284 |
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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. |
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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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
References: