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Millipore Technical Publications

Protocol: Immunohistochemical Methods to Detect Nitrotyrosine

Immunohistochemical Methods to Detect Nitrotyrosine

Tyrosine nitration has been shown to be commonly found in a large number of diseased tissues including amyotrophic lateral sclerosis (also known as Lou Gehrig’s disease), atherosclerotic arteries, lungs of septic and ARDS patients, multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease (1). Upstate Biotechnology’s anti-nitrotyrosine antibodies (catalog # 05-233 and 06-284) are suitable for use in a variety of applications such as immunoblotting (2), immunoprecipitation (2), enzyme-linked immunoadsorbent assays (ELISA), as well as immunohistochemistry and immunocytochemistry. Although generally more specific, monoclonal anti-nitrotyrosine demonstrates weaker immunoreactivity than the polyclonal antibody. The polyclonal anti-nitrotyrosine is therefore recommended for immunochemical studies, except when working with rabbit tissue.

Regardless of the application that anti-nitrotyrosine antibodies are used in, it is of great importance that the protocol used is optimized for the tissue and disease state of interest. The following immunohistochemistry protocols can be used as guidelines – antibody dilutions, incubation times, and blocking conditions may need to be adjusted according to the system studied. In addition to the actual experiment, controls that demonstrate the specificity of antibody binding should be run in parallel.

Antibody inhibition control

Specific binding of anti-nitrotyrosine to nitrated tyrosine residues can be inhibited by incubating the antibody with 3-nitrotyrosine (10 mM solution in PBS or TBS, adjust pH to 7.4 after adding nitrotyrosine; commercially available) or with nitrotyrosine-containing peptides (10 to 100 microM solution) for 1 hour at room temperature prior to applying the antibody to the tissue of interest (3).

By blocking the primary antibody, non-specific interactions, e.g. non-specific binding of the secondary antibody, can be demonstrated. In contrast, the antibody should not be inhibited by phosphotyrosine, methyltyrosine, dihydroxyphenylalanine, or aminotyrosine. All blocking solutions should be made up fresh and discarded if not used within one day.

Dithionite reduction

CAUTION: Dithionite is caustic and should be handled with extreme care. Wear gloves when handling! Dithionite is also highly reactive and very sensitive to air and moisture; store in small aliquots in a vacuum desiccator. Discard vial if powder does not flow freely.

Nitrotyrosine can be reduced to aminotyrosine in situ with dithionite (sodium hydrosulfite), which can no longer be bound by anti-nitrotyrosine antibodies. Dithionite solution (100 mM) can be prepared in 100 mM sodium borate, pH 9, in a sealed, degassed vacutainer tube and should be used within 1 hour of preparation. One milliliter of the solution is withdrawn with a syringe and the tissue section is washed three times with approximately 300 microliters each. Dithionite treatment can also be performed on proteins transferred onto immunoblotting membranes. Reoxidization of aminotyrosine to nitrotyrosine is possible as well, although the retrieved immunoreactivity is much weaker. This process is described in Viera et al., 1998.

Dithionite Reduction on Proteins Transferred onto Blotting Membranes

1. Perform the Immunoblotting assay as outlined in our certificate of analysis with the following exceptions: After blocking the membrane, prepare 10mM Nitrotyrosine in 3% dry milk in PBS with 0.05% Tween-20. Make sure the pH is 7.4. Co-incubate the membrane with the primary anti-Nitrotyrosine antibody for 2 hours at room temperature. Nitrotyrosine will block the specific antibody binding.
2. Repare fresh 0.1M Sodium Hydrosulfite (Dithionite) in 50 mM bicarbonate pH 9.0.
3. Incubate the membrane at room temperature in 10ml 0.1M Dithionite with agitation for approximately 1 minute, then wash and develop the blot as usual. The Dithionite will reduce the Nitrotyrosine to aminotyrosine. Therefore, the specific Nitrotyrosine antibody will not bind to Aminotyrosine.

Positive control

Tyrosine residues can be nitrated directly in situ on tissue sections using peroxynitrite (catalog # 20-107) to create a positive control. After the tissue section is deparaffinized, the slide is covered with a drop of PBS, pH 7.4 into which 10 microliters of peroxynitrite solution are mixed. Care should be taken that the solution maintains a pH of 7.4.

Blocking agents

Occasionally, commercially available proteins that are used to generate blocking solutions contain significant levels of nitrotyrosine. This leads to non-specific binding of the primary antibody and high overall background staining. Powdered milk (due to mastitis in milk cows), trypsin, and bovine serum albumin (BSA) have been shown to be endogenously nitrated. In general, the best solution to overcome this problem is to replace the nitrated blocking agent with protein from a different lot.

MacMillan-Crow et al., Proc. Natl. Acad. Sci. U.S.A. 93: 11853, 1996

Ye et al., Methods in Enzymology, ed. L. Packer, Academic Press, San Diego,
269:201 1996


General note: Where feasible, the solutions in the protocols below should be filtered through a 0.45 micron membrane prior to use or prepared with filtered PBS (150 mM NaCl, 10 mM potassium phosphate, pH 7.4). In initial experiments, a range of dilutions of the primary antibody of 1:50 to 1:1000 of a 1 mg/ml stock should be tested to determine the optimal antibody concentration. Incubations are generally performed in a humid chamber at room temperature unless otherwise noted. Often, overnight incubations can be reduced to 30 minutes to 3 hours at 37°C. The anti-nitrotyrosine antibody should be diluted in a solution that contains a protein for stabilization such as serum, milk, or BSA to prevent denaturation of the antibody sample.