M2 epifluorescence microscope

After transcardiac perfusion with 4% PFA in PBS, the eyes were removed, post-fixed with 4% PFA for 2 h, at room temperature, and cryoprotected in 30% sucrose overnight. Retinas were dissected out and washed extensively in PBS before blocking in staining buffer (10% normal goat serum and 2% Triton X-100 in PBS) for 30 min. RBPMS guinea pig antibody was custom made by ProSci Inc (Poway, CA, USA) and used at 1:4,000 as described before (Zhang et al., 2019b (link)). Floating retinas were incubated with primary antibodies overnight at 4°C and washed three times for 30 min each with PBS. Secondary antibodies (Cy3) were then applied (1:200–400; Jackson ImmunoResearch, West Grove, PA, USA) and incubated for 1 h at room temperature. Retinas were again washed three times for 30 min each with PBS before a coverslip was attached with Fluoromount-G (SouthernBiotech, Birmingham, Alabama). RGC were counted, 6–9 fields randomly sampled from peripheral regions of each retina using a 40× lens and a Zeiss M2 epifluorescence microscope, and RBPMS⁺ RGCs were counted by Volocity software (Quorum Technologies Inc., Puslinch, ON, Canada). The percentage of RGC survival was calculated as the ratio of surviving RGC numbers in injured eyes compared to contralateral uninjured eyes. The investigators who counted the cells were blinded to the treatment of the samples.

Whole-mount retinas were immunostained with the RBPMS antibody, 6–8 fields randomly sampled from peripheral regions of each retina using a 40X lens with a Zeiss M2 epifluorescence microscope, and RBPMS + RGCs counted by Volocity software (Quorum Technologies). The percentage of RGC survival was calculated as the ratio of surviving RGC numbers in injured eyes compared to contralateral uninjured eyes. The investigators who counted the cells were masked to the treatment of the samples.