Figure 2.

Immunohistochemical colocalization of a polyclonal anti–PGI₂ synthase Ab and an mAb against 3-nitrotyrosine in hypoxia-reoxygenated BCA. The yellow coloring resulting from a computer-generated overlay of green (3-nitrotyrosine) and red (PGI₂ synthase) fluorescence indicates areas of the colocalization of antinitrotyrosine and anti–PGI₂ synthase Abs in BCA with or without hypoxia–reoxygenation treatment. All pictures were obtained under 400-fold magnification with identical camera and print settings. (A) 3-nitrotyrosine staining in a sham-treated artery (green), where 3-nitrotyrosine staining is very weak and the endothelium is intact. The green wiggly line is due to endogenous fluorescence of the lamina and not specific immunostaining for 3-nitrotyrosine. (B) 3-nitrotyrosine staining in a hypoxia-reoxygenated artery; both endothelium and vascular smooth muscle cells are strongly immunopositive for 3-nitrotyrosine (green). (C) The staining of PGI₂ synthase Ab in a hypoxia-reoxygenated artery. Dense staining with anti–PGI₂ synthase Ab was visible in both endothelium and smooth muscle (red). (D) A computer-generated overlay of the stainings with the Abs against 3-nitrotyrosine (B) and PGI₂ synthase (C) in a hypoxia-reoxygenated artery. Yellow indicates the colocalization of the binding with both Abs. (E) An hypoxia-reoxygenated artery was stained for antinitrotyrosine Ab in the presence of 10 mM free 3-nitrotyrosine. Only the autofluorescence of the lamina is visible. (F) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of L-NMMA, where the staining for 3-nitrotyrosine is only weakly visible in both endothelium and vascular smooth muscle. (G) 3-nitrotyrosine stainings in a hypoxia-reoxygenated artery in the presence of PEG-SOD, where 3-nitrotyrosine staining is weakly visible in vascular smooth muscle. (H) An hypoxia-reoxygenated artery was stained for 3-nitrotyrosine when the Ab against 3-nitrotyrosine was omitted, where only the autofluorescence of the lamina is visible.