Retinal Vessel Density Analysis

Retinal flatmounts were prepared and the blood vessels were stained using fluorescein-conjugated isolectin B4 (IB4) (Table S1). Immunostaining with IB4 and DAPI was performed for two days at 4 °C. Following incubation with the antibodies, retinae were washed 3 times for 5 min each in phosphate buffer solution (PBS) and were embedded using aqua-poly/mount (18606-20; Polysciences Europe). For analysis, multiplane z-series were collected from fields of views of the vascular network from wildtype and KO mice by using 25× objective lens (VisiScope CSU-X1 confocal system; Visitron Systems). The series included the vitreal surface to the outer plexiform layer (OPL), and we captured at least 4 fluorescent images/retina/central and peripheral areas. Next, we performed a z-stack projection, dividing the z-series in two sets: the superficial vascular plexus (SVP) and the deep vascular plexus (DVP). The retinal vessel density was quantified using the angiotool software [32 (link)].