Fig. 10.

Double immunofluorescence staining for perlecan, 3-nitrotyrosine epitopes and CD14-positive cells in human atherosclerotic lesion sections. Frozen sections (5 μm) of human atherosclerotic lesions were incubated with monoclonal or polyclonal primary antibodies: anti-nitrotyrosine (rabbit IgG), anti-human perlecan (mouse mAb, clone 7B5, perlecan domain III) anti-human CD14 (mouse mAb), non-immune rabbit IgG and non-immune mouse IgG, and subsequently the following detection antibodies: goat anti-rabbit cyanine-3 (Cy-3)-labelled IgG or goat anti-mouse Cy-2-labeled IgG. DAPI was used to image cell nuclei. Images were acquired as described in the Materials and Methods. A) Heavily thickened intima of an artery with a type III/IV lesion. Epitopes for 3-nitrotyrosine (red signal) show marked co-localization with perlecan (green) present in the basement membrane of the endothelium (arrow) as well as with those of the vasa vasorum (dotted arrow). B) The signal for 3-nitrotyrosine (red) was frequently co-localized with CD14-positive cells (macrophages, green). The red signal for nitrotyrosine underneath the bulk of macrophages results from multiple oblique sections through a vas vasorum. C) In sections where the antibodies for perlecan/CD14 and for 3-nitrotyrosine were replaced with non-immune mouse IgG (control, green) and non-immune rabbit IgG (control, red), no staining in the intima was observed. The faint background staining in the media (also observed when no primary or secondary antibodies were applied to the sections) results from elastic membranes located in this area. Scale as indicated in the bottom images.