After the retinas were embedded in paraffin, retinal sections of 4 µm thickness were prepared. Three percent hydrogen peroxide was used to inactivate endogenous peroxidases, and EDTA, adjusted to pH 9.0, was used for heat-induced antigen retrieval for 8–10 min. After rinsing with PBS (1X; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), retinal sections were incubated with rabbit anti-cleaved caspase-3 primary antibody (#9661s, dilution: 1/300, Cell Signaling Technology, Danvers, MA) was performed at 4 °C overnight, followed by incubation with goat anti-rabbit immunoglobulin G (IgG; PV-9001, ZSGB-BIO, Beijing, China) secondary antibody at 37 °C for 1 h. After washing, 3,3′-diaminobenzidine (DAB, ZLI-9017, ZSGB-BIO) was used to visualize immunoreactivity. All sections were then counterstained with hematoxylin to stain the nuclei. Finally, sections were dehydrated and mounted for microscopic examination.
For the TUNEL assay, retinal sections were deparaffinized and rehydrated. After rinsing with PBS, the sections were treated with proteinase K (Roche Applied Science, Indianapolis, IN) and quenched with 3% hydrogen peroxide; then they were incubated in a terminal deoxynucleotidyl transferase (TdT) reaction mix (Roche Applied Science) for 1 h at 37 °C. Sections were then incubated for 5 min with 4',6-diamidino-2-phenylindole (DAPI) at room temperature.
For the TUNEL assay, retinal sections were deparaffinized and rehydrated. After rinsing with PBS, the sections were treated with proteinase K (Roche Applied Science, Indianapolis, IN) and quenched with 3% hydrogen peroxide; then they were incubated in a terminal deoxynucleotidyl transferase (TdT) reaction mix (Roche Applied Science) for 1 h at 37 °C. Sections were then incubated for 5 min with 4',6-diamidino-2-phenylindole (DAPI) at room temperature.