Immunohistochemistry Protocol for Retina Analysis

The protocol for immunohistochemistry as previously described.⁴⁷ The retinas were incubated in PBS with 1% Triton X-100 and 0.5% Tween 20 for 1 h at room temperature and in 4% BSA for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies: RBPMS (1:500, Abcam), ChAT (1:100, Abcam), VGlut2 (1:100, Frontier Institute), syntaxin (1:100, Abcam), tyrosine hydroxylase (1:100, Abcam) and Prox1 (1:100, AngioBio) in blocking buffer. Secondary anti-rabbit, mouse, and goat IgG, conjugated with Alexa TM488, TM594, and 633, respectively (1:1,000; Molecular Probes), were applied for 1 h at room temperature. The retinas then were flat mounted, and the sections were mounted on slide glass. The TUNEL assay was performed based on our previous reports.⁴⁸ (link)^,49