The eyeballs were collected and fixed, and then the retinas were dissected, washed, blocked, and permeabilized, as previously described.
24 The retinas were incubated with Alexa Fluor 594-conjugated Isolectin B4 (1:200; ThermoFisher Scientific, Waltham, MA, USA) or primary antibodies against GFAP (1:500, Z033401-2; Agilent Technologies, Santa Clara, CA, USA), CD206 (1:200, 141710; BioLegend, San Diego, CA, USA), Iba1 (1:200, 019-19741; FUJIFILM Wako Chemicals, Richmond, VA, USA), and RBPMS (1:200, ABN1362; MilliporeSigma, Burlington, MA, USA) at 4°C overnight. After rinsing, the retinas were incubated with appropriate Alexa Fluor 488-conjugated secondary antibodies (1:400; ThermoFisher Scientific) for 4 hours at 4°C. Last, the retinas were mounted and images were obtained using a confocal microscope (LSM 800; Carl Zeiss, Inc., Thornwood, NY, USA). To quantify central avascular area and neovascular tufts of the retina, the surface areas of the total retina, central avascular zone, and neovascularization were measured using ImageJ software (Bethesda, MD, USA), as previously described.
25,26 The avascular area and neovascularization were expressed as a percentage of the total retinal surface area. For cell counting, eight images were taken at the peripheral region of each retinal flatmounts, and cells were manually counted and averaged for each sample.