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Figure 5 Immunohistochemical detection of nitrated prostacyclin-synthase (PGI2-S) in slices of aortic rings from rats with and without nitroglycerin treatment. In tissue from sham-treated rats, nitration was virtually absent (A), PGI2-S expression was not significantly modified (B), and hence, the overlay of both only yielded marginal background staining (C). In tolerant tissue, nitrated protein gave a clear positive signal (D), the amount of PGI2-S was comparable to control tissue (E), and the overlay resulted in a deep yellow staining for co-localization of PGI2-S and nitration (F). The specificity of the 3-nitrotyrosine antibody in tolerant tissue was confirmed by three control experiments: 1) the antibody was blocked by co-incubation with authentic 3-nitrotyrosine (G); 2) protein-bound 3-nitrotyrosine was reduced by sodium dithionite before antibody incubation (H); and 3) only the secondary antibody was used (I). Green fluorescence (Alexa 488-labeled secondary antibody) corresponds to nitrated protein; red fluorescence (Alexa 568-labeled secondary antibody) to PGI2-S; and yellow to the computer-generated overlay of both stainings and, accordingly, to nitrated PGI2-S.
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