Figure 4

Identification of leukocyte subtypes in stage II remodeling vessels. Immunostaining for (A and D) CD45, (B and E) CD56 (uNK cells), and (C and F) CD68 (macrophages) revealed that leukocytes infiltrating remodeling vessels are uNK cells and macrophages (arrowheads). C: Inset– representative negative control. Serial immunostaining (A) for (G–I) matrix metalloproteinases (MMPs) revealed that infiltrating leukocytes express (G) MMP-9 (arrowheads) and (H) MMP-7 (arrowheads), but (I) not MMP-2. J–M: Dual immunofluorescence confirmed that uNK cells and macrophages produce MMPs: (J) cell surface CD56 (red) and cytoplasmic MMP-9 (green) co-localized in cells in the vascular wall of remodeling vessels, (K) CD56 (red) and MMP-7 (green) also co-localized as did (L) CD68 (red) and MMP-9 (green) and (M) CD68 (red) and MMP-7 (green) (arrowheads). N: In situ zymography revealed gelatinase activity in leukocytes infiltrating remodeling vessels, (O) High magnification of (N). P–R: Negative controls: (P) Primary and secondary antibody was omitted to control for autofluorescence, (Q) the first secondary antibody was omitted to control for cross-reaction of the first primary and second secondary antibodies and, (R) mouse IgGs were applied to negative control sections to control for non-specific staining. Scale bars: 20 μm (J–R), 25 μm (A–C and G–I), and 50 μm (D–F).