Tdt reaction mix

At 1, 3, and 7 d of reperfusion, 5 rats from each group were randomly chosen. The preparation of paraffin sections of brains was described above. A 50 μl volume of TdT reaction mix (including 45 μl Equilibration Buffer plus 5 μl Nucleotide Mix and 1 μl rTdT enzyme) (Promega Corporation, Wisconsin, USA) was added followed by incubation at 37°C for 1 h in the dark. Then, the slices were stained with DAPI at 37°C for 10 min; neutral balsam was added as the sealing agent in a dark room. The cerebral cortex was observed (×400 magnification) and photographed using a fluorescence microscope (DM5000B, Leica Microsystems Inc., Germany). Three random fields were chosen in each slice. An investigator blinded to the experimental design analysed the slides. The total number of cells and the number of TUNEL-positive cells were counted to evaluate the neuronal cell apoptosis rate.
Apoptosis rate = number of TUNEL-positive cells/total number of cells × 100%.

Slides were placed on the Leica Bond RX IHC stainer. All steps besides dehydration, clearing and cover slipping were performed on the Bond RX in TPSR. Slides were deparaffinized. Antigen retrieval was performed on the Bond RX using Triton X-100 (Cat#T9284, St. Louis, MO) for 5 min. Slides were incubated with Equilibration Buffer (#G7130, Promega, Madison, WI) for 5 min, followed with the TdT reaction mix (#G7130, Promega, Madison, WI) for 10 min, and SSC-x20 (#G7130, Promega, Madison, WI) for 10 min. The Bond Intense R detection system (#DS9263, Leica, Buffalo Grove, IL) was used for visualization. Slides were dehydrated, cleared and cover slipped. A defined surface area of 8.15 mm² that contained approximately 50–60 seminiferous tubules from each section was randomly selected for analysis of the TUNEL assay. Positive cell detection and counting was performed using QuPath open-source software for digital image analysis (Bankhead et al., 2017 (link)). Two serial sections per sample were analyzed and the positive cell counting results (as a percentage of total cells counted) were recorded.