Fluorescent mounting media

TUNEL assay was performed to evaluate the degree of apoptosis in tissue and cells. Following fixation with 4% paraformaldehyde at room temperature for 30 min, tissue sections or cells were incubated with proteinase K at room temperature for 15 min, placed in 3% H₂O₂ for 15 min at room temperature and treated using a TUNEL detection kit for 60 min at 37°C. Following incubation, the PBS-rinsed cells were co-labeled with 1 µg/ml DAPI working solution for 10 min at 37°C. The positive cells were mounted with fluorescent mounting media (Beijing Solarbio Science & Technology Co., Ltd.) and analyzed using ImageJ 1.8.0 software (National Institutes of Health). More than 10 fields of view/section for each sample were assessed. The labeled cells were visualized using an Olympus BX53 fluorescence microscope (magnification, ×100; Olympus Corporation).

TUNEL assay was used to detect apoptotic cells using a TUNEL kit (cat. no. C1082; Beyotime Institute of Biotechnology), according to the manufacturer's protocol. HPMVECs were fixed with 4% paraformaldehyde at room temperature for 15 min and permeated with 0.1% Triton X-100 for 5 min at room temperature. Subsequently, cells were incubated with TUNEL at 37˚C for 1 h and cell nuclei were counterstained with DAPI (10 mg/ml; Koritai Biotechnology) for 5 min at room temperature. A total of five fields of view were randomly selected from each slice. The number of positive cells was mounted with fluorescent mounting media (Beijing Solarbio Science & Technology Co., Ltd.) and the observation was conducted under a fluorescence microscope (Nikon Corporation). The cell apoptosis rate (%) was calculated as follows: Number of apoptotic positive cells/total number of cells.

The number of apoptotic cells was assessed using an apoptosis detection kit (Roche Diagnostics) in accordance with the manufacturer's protocols. Briefly, the cells were fixed in 4% paraformaldehyde at 37˚C for 15 min and incubated with proteinase K (Beyotime Institute of Biotechnology) at room temperature for 15 min. Subsequently, the cells were placed in 3% H₂O₂ at room temperature for 15 min, cultivated with TUNEL regent (Beyotime Institute of Biotechnology) at 37˚C for 1 h and counterstained with DAPI for 5 min at room temperature. The number of positive cells was mounted with fluorescent mounting media (Beijing Solarbio Science & Technology Co., Ltd.) and analyzed using ImageJ 1.8.0 software (National Institutes of Health). Overall, >10 fields of view/section for each sample were assessed. A fluorescence microscope was used for the visualization of the positive cells.