Tunel detection kit

TUNEL method was used to detect renal tissue apoptosis. According to the manufacturer’s instructions, use TUNEL detection kit (Servicebio, Wuhan, China) to detect 4 um tissue sections. First, the paraffin-embedded tissue sections were deparaffinized and transparent, and then the sections were repaired with proteinase K working solution for 30 min, and 0.1% triton was added dropwise to the sections and incubated at room temperature for 20 min. Then, the sections were incubated in a mixed buffer containing TDT enzyme-dUTP at 37°C for 2 h. DAPI staining solution was added dropwise and incubated at room temperature in the dark for 10 min. The sections were washed with PBS, and finally mounted with anti-fluorescence quenching mounting tablets, and the sections were observed through a fluorescence microscope to estimate the number of TUNEL-positive staining cells.

The deparaffinized sections of quadriceps were stained with TUNEL detection kit (GDP1042, Servicebio, Wuhan, China). Then, the slides were observed under a fluorescence microscope, and images were captured with digital camera (BX51, Olympus, Japan). The apoptotic index (positive cells / total cells × 100%) was calculated from five randomly visions in each section.

MSCs were cultured on cell slides until reaching 80% confluence and then replaced with serum-free medium for 24 hours. Then, they were fixed with 4% PFA for 30 minutes and washed 3 times with PBS. Then, we followed the manufacturer's instructions of TUNEL detection kit (Servicebio, China) and took pictures under a fluorescence microscope. The red light represented the apoptotic cell nucleus stained by TUNEL, and the blue light was the cells stained by DAPI. The percentage of the number of red fluorescent cells to the number of blue fluorescent cells represented the proportion of apoptotic cells in the total cells.