VEGFC mediates hypoxia-induced retinal neovascularization. A. After exposure to 75% oxygen from P7 to P12, pups were returned to room air to develop the relative hypoxia. Pups were administered intravitreally with 1 μg/0.5 μl/eye of control or VEGFC siRNA at P12 and P13 and at P15 the retinas were isolated and either tissue extracts were prepared and analyzed for VEGFC levels by Western blotting using its specific antibody and normalized to β-tubulin or fixed, cross-sections made and stained by immunofluorescence for CD31 and Ki67. The right column shows the higher magnification (40 ×) of the areas selected by rectangular boxes in the left column images. B. Retinal EC proliferation was measured by counting CD31- and Ki67-positive cells that extended anterior to the inner limiting membrane per section (n = 6 eyes, 3 sections/eye). C. All the conditions were the same as in panel A except that control or VEGFC siRNAs were injected intravitreally at P12, P13, and P15 and at P17 the retinas were isolated, stained with isolectin B4, flat mounts were made and examined for EC filopodia formation at 40 × magnification. D. All the conditions were the same as in panel A except that the sections were stained for CD31, VEGFR3 and DAPI. E. All the conditions were the same as in panel C except that retinal neovascularization was measured at 2.5 × magnification. Retinal vascularization is shown in the first column. Neovascularization is highlighted in red in the second column. The third row shows the selected rectangular areas of the images in the first column under 10 × magnification. F & G. Retinal neovascularization (F) and avascular area (G) were determined as described in “Materials and Methods.” The bar graphs represent quantitative analysis of three blots or 6 retinas. The values are presented as Mean ± SD. * p < 0.01 vs normoxia + control siRNA; ** p < 0.01 vs hypoxia + control siRNA. Scale bar represents 50 μm and 20 μm in panel A, far left column and far right column, respectively, 20 μm in panels C & D, 300 μm and 50 μm in panel E, far left column and far right column, respectively.