Quantifying RGC Survival Post-NMDA Insult

RGC survival was estimated 3 weeks after NMDA injection on retinal flat-mounts^{2 (link),37 (link),66 (link)}. In brief, flat-mounted retinae were washed with 0.1 M PBS and permeabilized with 100% methanol for 20 min at −20 °C. Retinal flat-mounts were immunofluorescently labeled for β3tubulin, a marker of RGCs. In this aim, retinae were incubated with a rabbit anti-β3tubulin antibody (1:500, Abcam, Cambridge, UK, #ab18207) diluted in blocking solution (PBS containing 5% bovine serum albumin (BSA), 0.3% Triton X-100, and 0.05% sodium azide) and then with a secondary anti-rabbit antibody. Images were taken with a Leica SPE-II confocal microscope at ×40. RGCs were counted in regions of 62 500 µm² at 1 and 1.5 mm from the optic disk in the four retinal quadrants.