One step tunel assay kit

To detect cell apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed using the One-Step TUNEL Assay Kit (Beyotime, China) at 24 h after SAH. Briefly, brain specimens were immersed in a TUNEL mixture for 2 h at 37.5°C, followed by a stained with DAPI. The data were expressed and analyzed in the same procedure as for FJC staining.

One-step TUNEL assay kit (Beyotime, Shanghai, China) was used to detect apoptosis according to the standard protocol. Paraffin-embedded sections were incubated with proteinase K for 30min at 37°C. Next, each slice was incubated with 50μL TUNEL mixture for 1 h at 37°C in the dark. The cells with green fluorescence were defined as apoptotic cells using fluorescence microscopy (Nikon Eclipse 80i, Japan) at 200× magnification. At least 4 areas were selected for each slide.

TUNEL labeling was performed before antibody incubation procedure with a one step TUNEL assay kit (Beyotime, C1089). Specimens were incubated in detection buffer at room temperature for one hour, followed by three times washing in PBS.

To detect neuronal cell death, double immunostaining of NeuN and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out using One Step TUNEL Assay Kit (Beyotime, China). Briefly, brain slices were incubated with anti-NeuN (ab177487, 1:200, Abcam, USA) overnight at 4 °C, and then immersed in TUNEL mixture at room temperature for 1 h, followed by DAPI staining. The TUNEL positive neurons were counted in 3 microscopic fields at 400 × magnification at the left, right, and bottom of peri-infarct area in each mouse by a blinded observer. Data were expressed as the ratio of TUNEL positive neurons (relative to Sham group).

The cell nuclei were stained with DAPI following the instructions of the One-Step TUNEL Assay Kit (Beyotime, Nanjing, China) and then photographed with a fluorescent microscope. The number of DAPI-positive cells and total cell number were counted, and their ratio equals the apoptosis rate [14 (link)].

Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was conducted to precisely detect cells experiencing programmed cell death (PCD) with the one-step TUNEL Assay kit (Beyotime, China) following the manufacturer’s instructions. Briefly, tissue sections were twice dewaxed in xylene for 5-10 minutes. Then tissues were immersed into anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 2 minutes. Tissues were treated with DNase-free proteinase K (Beyotime, 20μg/mL) at room temperature for 15-30 minutes and then washed with PBS for three times to remove excess proteinase K. Afterwards, the labelling solution containing terminal deoxynucleotidyl transferase, buffer, and fluorescein dUTP was added to tissues for labelling reaction at 37 °C for 60 minutes in a humidity chamber. Following incubation, excess labelling solution was washed off smears with PBS, and then cell smears were mounted with fluorescent microscopy mounting solution. Images were captured and analyzed using a CCD camera (Olympus, Japan).

For a terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) assay, we followed standard protocols using a One-Step TUNEL Assay Kit (Beyotime, China). Briefly, ocular tissues were fixed with 4% paraformaldehyde, rinsed with PBS, and permeabilized with 0.5% Triton X-100 to obtain the fragmented DNA of apoptotic cells. Then, TUNEL detection solution was added to each sample, and the samples were incubated for 60 min at 37°C in the dark. Finally, the sections were rinsed with PBS again and costained with DAPI (4,6-diamidino-2-phenylindole).