Bond rx ihc stainer

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.

Immune cell infiltration was detected by anti-myeloperoxidase (MPO) staining performed at the VUMC Translational Pathology Shared Resource. Briefly, slides were placed on the Leica Bond RX IHC stainer. Slides were placed in a Protein Block (DAKO) for 10 minutes, then incubated with anti-MPO (DAKO A0398) for one hour at 1:4000 dilution. The Bond Polymer Refine detection system was used for visualization. Slides were then dehydrated, cleared and coverslipped. To quantify immune cell infiltrates, a single mucosal layer was visualized in a 20x field of view (FOV) at a time, and the number of MPO⁺ cells were counted per FOV in that region. The number of MPO⁺ cells was quantified in three FOVs per mouse per treatment. The average MPO⁺ cells per mouse per treatment (n = 3) were plotted using ggplot2 and ggprism, and differences were tested using one-way ANOVA and Tukey’s HSD test in R v. 4.0.3. Presented images were captured using a Leica SCN400 Slide Scanner at the VUMC DHSR as described above.