St533

Samples of hDNM and pDNM (5 mg each) were digested in proteinase K solution (200 μL, 1 mg/mL; ST533, Beyotime Biotechnology) at 56°C until digestion was complete. After centrifugation at 10,190 × g for 10 minutes, the DNA content of the supernatant was determined using a Quant-iT PicoGreen kit (P11496, Invitrogen, Carlsbad, CA, USA) according to the YY/T 0606.25-2014 standard.^{21 (link)}

An In Situ Cell Death Detection Kit (Roche, 11684817910, Basel, Switzerland) was used to detect apoptotic DNA fragments. Paraffin sections (5 µm) of the brain were obtained 24 hours after the above HIBD. After routine deparaffinization and hydration, the brain tissue was incubated with 20 µg/mL proteinase K working solution (ST533, Beyotime) at 37°C for 30 minutes and washed with PBS, and the area around the sample was dried. The sample and TdT-mediated dUTP nick-end labeling (TUNEL) reaction mixture were incubated in a dark and humid environment at 37°C for 1 hour and then washed three times with PBS (pH 7.4); the plate was then mounted with an anti-fluorescence quencher containing DAPI. A fluorescence microscope was used to examine apoptosis. ImageJ was used to measure the number of TUNEL-positive cells and calculate the apoptotic index (the ratio of the number of apoptotic cells to the total number of cells in a field of view).