TUNEL Assay for Retinal Apoptosis

To detect cells undergoing apoptosis in retinal capillaries, terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay was performed using a kit (ApopTag Fluorescein In Situ Apoptosis Detection; Millipore Sigma) as described previously [39 (link)]. Briefly, RTDs were fixed in paraformaldehyde, permeabilized in a pre-cooled mixture of a 2:1 ratio of ethanol/acetic acid, washed in PBS, exposed to equilibration buffer, and incubated for 1 h with deoxyribonucleotidyl transferase (TdT) enzyme in a moist chamber at 37 °C. Following incubation, RTDs were exposed to anti-digoxigenin peroxidase, washed in PBS, counterstained with DAPI, and mounted using anti-fade reagent (SlowFade Diamond Antifade, Cat#S36963; Invitrogen, Carlsbad, CA, USA). At least five images representing random fields of the RTD slide were captured using a digital microscope (Nikon Eclipse; TE2000-S) and TUNEL-positive cells per total number of cells per field were analyzed.