TUNEL Assay for Corneal Apoptosis Detection

The One-step TUNEL Apoptosis Assay Kit (Beyotime, Beijing, China) was used to perform the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay) for 8-μm-thick corneal cryosections. Briefly, cryosections of the cornea samples were fixed with 4% paraformaldehyde for 60 min, and then washed thrice with PBS, followed by the addition of 0.3% Triton X-100 and incubation at room temperature for 5 min. Next, for the TUNEL staining, the TUNEL test solution was prepared according to the manufacturer’s instructions. To each sample, 50 μL of TUNEL solution was added and this incubated at 37 °C for 60 min in darkness. After rinsing three times with PBS for corneas, 4ʹ,6-diamidino-2-phenylindole (DAPI) was added to counterstain and seal each cryosection sample. Three cryosections from each rat’s cornea were analyzed and photographed by confocal laser scanning microscopy (CLSM; Zeiss LSM 800, Germany).