Eyes were enucleated from mice and fixed in 4% PFA in PBS for 1.5 h at room temperature and kept in methanol at −20 °C for further processing. The eyes were rehydrated in PBS for 1 h at room temperature. Connective tissues and extraocular muscles attached to the back of the eyecup were removed by using scissors and forceps under a microscope. A 28-gauge needle was then used to make a small hole in the ora serrata and the cornea and lens were removed by cutting along the ora serrata using scissors. Retinas were carefully detached from the eyecups and blocked with a blocking buffer (50% FBS, 20% goat serum (GS), and 0.1% Triton X-100). The retinas were then incubated with anti-collagen IV (AB756P, Millipore) diluted 1:250 in a blocking buffer (20% FBS, 20% GS, and 0.1% Triton X-100) at 4 °C overnight. The retinas were then rinsed with PBS three times for 10 min each and incubated with appropriate fluorescent-conjugated secondary antibody (1:1000, Jackson ImmunoResearch) for 1 h at room temperature. The hyaloid vessels, which were seen with collagen IV staining, were removed using forceps under fluorescence illumination with a stereomicroscope (SMZ25, Nikon, Japan). The retinas were flattened by four radial cuts and mounted on a glass slide with Fluoromount-G mounting solution (0100-01; SouthernBiotech) and photographed by fluorescent microscopy. Vitreous neovascularization on P17 was quantified by using the ImageJ plugin SWIFT_NV developed by Stahl et al. for ImageJ [93 (link)] and presented as percentages of the total retinal area, which was measured by ImageJ.
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