TUNEL Assay for Apoptosis Detection

TUNEL staining was conducted as described in a previous study [34 (link)]. In brief, liver tissues of septic mice were fixed, embedded, and sectioned into 4 μm thick slices. The Apoptosis Assay Kit (Beyotime) was used to detect apoptotic cells in liver tissue sections according to the manufacturer's instruction. The liver slices were deparaffinized, rehydrated with graded ethanol dilutions, incubated with 20 μg/ml Proteinase K (Beyotime) at 37°C for 30 min, and washed with PBS three times. Then, the slices were incubated with 50 μl TUNEL working solution at 37°C for 60 min, followed by incubation with DAPI (Beyotime) for 15 min protected from light. The slices were washed with PBS three times, and the fluorescence signals (488 nm excitation wavelength) were observed by fluorescence microscopy. For statistical analysis, three fields under ×200 magnification were observed and TUNEL-positive cells were counted, as previously described [35 (link)].