Time course of expression of collagen type IV, VEGF, and MMP-9 in the ischemic retina. Ischemia-induced retinal neovascularization was initiated and retinas subjected to hyperoxia were obtained at 0, 1, 2, 3, and 5 days (P12, P13, P14, P15, and P17) after their return to normoxic conditions. A: Retinas were co-stained with a polyclonal antibody directed to collagen type IV (green) and biotinylated mAb HUIV26 (red). B: Retinas were co-stained with a polyclonal antibody directed to MMP-9 (green) and a mAb directed to VEGF (red). C: Retinas were co-stained with a polyclonal antibody directed to MMP-9 (green) and biotinylated mAb HUIV26 (red). D: Retinas were co-stained with a polyclonal antibody directed to MMP-2 (green) and biotinylated mAb HUIV26 (red). Flat mount retinas were analyzed on a Nikon TE300 microscope with epifluoresence filters and images were captured with a charge-couple device camera for analysis. Co-localization is indicated in yellow. White bars equal 50 μm. E: Quantitation of the number of HUIV26 cryptic sites (spots) per retina. The total number of HUIV26 cryptic sites (spots) was manually counted within the total area of each retina and 18 retinas were counted per time point. Data bars represent the mean ± SD.