Quantification of Apoptotic Retinal Cells via TUNEL Assay

In situ cell death detection kit (Roche) based on labeling of DNA strand breaks [(TdT-mediated dUTP-X nick end labeling (TUNEL)] was used to quantify apoptotic retinal cells (Table 1, Additional file 1: Table S1). Briefly, the DNA cleavage can be detected by labeling the free 3′-OH termini with fluorescein modified nucleotides in an enzymatic reaction. According to the manufacturer’s protocol, retinal cell death detection was conducted using 14 µm retinal cryosections from 4% PFA (w/v) fixed eyes (methods described before). After three washes with PB, slides were incubated in phosphate buffered saline (PBS) with 1% Triton X-100 (v/v) for 5–10 min at RT in humidity chamber. Then, TUNEL mix reagent was incubated for 1 h at 37 °C in dark conditions. Thereafter, the reaction was stopped with three PB washes for 5 min at RT in the dark. Finally, preparations were mounted using Citifluor and coverslipped.