Figure 3

Induction of revascularization after hindlimb ischemia. (a) Quantification of microvessel density on hematoxylin-eosin frozen sections. Histograms represent mean vessel number per field of hematoxylin-eosin cryosections counted by two independent observers, 10 days after hindlimb ischemia. Results are expressed as mean ± SEM. *P < 0.05 versus PBS. (b) The presence of inflammatory cells such as polynuclear neutrophils (indicated with black arrow) and erythrocytes in the lumen of microvessels revealed by a hematoxylin-eosin staining indicate the functionality of blood microvessels. Bar = 10 µm, magnification inset × 400. (c) Left panel: Endothelial cells were identified by immunohistochemistry with anti-CD31 antibody revealed with Alexa fluor 555-secondary antibody (red) and vascular smooth muscle cells were immunolabeled with anti-smooth muscle actin (SMA) antibody revealed with Alexa fluor 488-secondary antibody (green). Nuclei were stained with DAPI (blue). Magnification: × 200. Right panel: Cells derived from endothelial progenitor cells were identified in the intima of microvessels in samples treated with MP loaded RANTES (10 nM) by immunohistochemistry with anti-CD34 antibody revealed with Alexa fluor 555-secondary antibody (red) and with anti-vWF antibodies revealed with Alexa fluor 488-secondary antibody (green). Nuclei were stained with DAPI (blue). Magnification: × 200, inset × 400. (d) Upper panel: Monocytes-macrophages were identified with anti-MOMA-2 antibody followed by HRP-labeled secondary antibodies and DAB with nuclei stained in blue with hemalum indicated with black arrows in high insert view. Bar: 1 mm (Upper left panel), Magnification: × 200, inset × 400 (Upper right panel). Middle panel: Negative control of monocytes-macrophages immunostaining. Slides were incubated with specific isotype instead of anti-MOMA-2 antibody, then with HRP-labeled secondary antibodies and DAB, and nuclei were stained in blue with hemalum. Bar: 1 mm (Middle left panel), Magnification: × 200, inset × 400 (Middle right panel). Lower panel: Histogram represents infiltrate cell area evidenced by anti-MOMA-2 immunostaining. Results are expressed as mean ± SEM of the ratio between infiltrated cells surface area and the total surface area of the section.