Apoptosis assay, TUNEL

After enucleating the eyes, a small incision was made into the cornea for faster penetration of the fixative, and the tissue was submerged in 4% PFA for 4 h. After rinsing the tissue, the whole eye was cryoprotected in a gradient of sucrose (15%, 20%, and 30%) and embedded in OCT. 40 μm sections of the central retina were permeabilized with a 0.1% Triton X-100 and 0.1% sodium citrate solution for 5 min at 4ºC, washed 4X for 5 min each, and incubated for 2 h at 37ºC with the TUNEL reaction solution following the manufacturer instruction. Following the TUNEL assay, sections were rinsed and blocked with 10% normal donkey serum and incubated overnight with primary S-opsin antibody. Corresponding secondary antibody (Alexa Fluor-488) was applied the next day and incubated for 3 h. To confirm specificity of the primary antibodies, control samples in which the primary antibody was omitted were included.