Apoptosis Detection in Retinal Capillaries

To identify cells undergoing apoptosis, terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) assay was performed using a commercially available kit (ApopTag, Sigma, Temecula, CA, USA) as previously described [47 (link)]. Briefly, retinal capillaries were fixed in paraformaldehyde, permeabilized in EtOH-acetic acid, washed in PBS, and incubated in deoxyribonucleotidyl transferase enzyme for one hour at 37 °C. The capillary networks were mounted using antifade reagent (SlowFade Diamond Antifade, Cat#S36963; Invitrogen, Carlsbad, CA, USA) and DAPI. Images from 10 random fields were captured using a digital microscope (Nikon Eclipse; TE2000-S, Nikon, Tokyo, Japan) and analyzed for TUNEL-positive cells.