Protease k without dnase

Paraffin sections of retinal tissue were routinely dewaxed to water. Immunohistochemistry pen was used to circle around the retinal tissue. 20 μg/ml protease K without DNase (st532, Beyotime, China) was dripped. The cells were incubated at room temperature for 30 min and washed with PBS for 5 min × 3 times. Prepare the TUNEL detection solution (TDT enzyme: fluorescent labeling solution = 1:9), add 20 μL to each retina, put the slices into a wet box containing water, incubate them at 37°C for 60 min in dark, and wash them with PBS for 5 min × 3 times. Each tissue was incubated with 10 μl DAPI at room temperature for 3 min, washed with PBS for 5 min × 3 times, sealed with anti-fluorescence quenching solution, observed and took photos under fluorescence microscope.

Retina and ARPE-19 cell apoptosis levels were detected using the One-Step TUNEL Apoptosis Assay Kit, according to the procedures from the manufacturer (C1089, Beyotime, China). According to the manufacturer’s instructions, paraffin-embedded eyeballs were dewaxed in xylene (5–10 min). Next, xylene was replaced, and dewaxing was performed for 5–10 min in the following sequence: anhydrous ethanol (5 min), 90% ethanol (2 min), 70% ethanol (2 min), and distilled water (2 min). The cells were fixed with 4% paraformaldehyde (G1101, Servicebio, China) for 30 min. Then, 20 μg/mL of protease K without DNase (ST532, Beyotime, China), immunostaining washing solution (P0106, Beyotime, China), and 50 μL of TUNEL test solution were added for 60 min in the dark.

Rats were deeply anesthetized by intraperitoneal injection of 10% chloral hydrate (300 mg/kg, vide supra), and then their heads were severed. The brains were cut in half according to the ischemic core. Half was used for Western Blotting assay. Half was fixed in 4% paraformaldehyde for 4 days. Then the brain tissue was embedded in paraffin and sectioned into 4 µm slices. Five slices in the ischemic core were obtained. The sections were dehydrated in alcohol gradients and incubated with 20 μg/ml protease K without DNase (Beyotime Biotechnology, Shanghai, China) at 37°C for 30 minutes. Subsequently, the samples were washed with PBS three times and were then ready for TUNEL staining (according to the procedure of TUNEL kit, Beyotime Biotechnology, Shanghai, China). Slides were re‐stained with 4’,6‐diamidino‐2‐phenylindole (DAPI) (Beyotime Biotechnology, Shanghai, China) for 10 minutes, and imaged under a fluorescence microscope to evaluate the degree of apoptosis. Five random images in one slice were obtained and the mean TUNEL positive cell number from five images was calculated as the TUNEL positive cell number of one rat. The images were obtained and analyzed by a staff blinded to the group setting.