TUNEL assay

A POD kit (cat. no. 11684817910; Roche Diagnostics) was used to measure TUNEL-positive apoptotic cells in tissue sections. Kidneys were harvested post-mortem and fixed in 4% paraformaldehyde at 4°C overnight, embedded in paraffin and cut into 5-µm thick sections. Renal paraffin sections (5-µm) were de-waxed and incubated with protease K. Subsequently, the sections were treated with the TUNEL reaction mixture and incubated for 60 min in a water bath at 37°C. DAPI (Beyotime Institute of Biotechnology) was used to stain nuclei. Images were obtained using a fluorescence microscope (magnification, ×20). Five fields of vision were randomly selected for each slice.