Peroxidase and alkaline phosphatase blocking reagent

C57Bl/6 males (11 weeks of age) were euthanized as per the guidelines of the University of Virginia Institutional Animal Care and Use Committee. Testes were harvested and fixed overnight in Bouin’s fixative (Sigma), followed by embedding in paraffin (Hogarth and Griswold. 2013 (link)). Five-micron-thick sections were deparaffinized in xylene, and rehydrated in a graded series of ethanol baths. Immunohistochemistry was performed on a robotic platform (Autostainer, Dako, Glostrup, Denmark). Endogenous peroxidases were blocked using Peroxidase and Alkaline Phosphatase Blocking Reagent (Dako). No antigen retrieval step was performed. Guinea pig anti-mouse SP-10 primary antibodies (B, C, and D) were used at a 1:1000 dilution. Goat anti-guinea pig-HRP conjugated secondary antibodies (Peroxidase-AffiniPure Goat Anti-Guinea Pig IgG (H+L) Code: 106-035-003, Jackson Immuno Research Laboratories) were used at a 1:200 dilution. The antigen-antibody reaction was assessed by incubation with 3,3’-diaminobenzidinetetrahydrochloride (DAB+) chromogen (Dako), as per the manufacturer’s instructions. All the slides were counterstained with hematoxylin, and were then dehydrated, cleared, and mounted for assessment and imaging. Periodic acid-Schiff histology was performed using a PAS kit (Sigma-Aldrich), following the instructions provided by the manufacturer.

TMAs were stained by IHC for immune cells and markers of immune activation and inhibition (Supplementary Table I). Slides were deparaffinized, and antigen retrieval was performed in the PT Link instrument (Dako, Glostrup, Denmark) in antigen retrieval solution at 97°C for 20 minutes. IHC was performed on a robotic platform (Autostainer, Dako). Endogenous peroxidases were blocked using peroxidase and alkaline phosphatase blocking reagent (Dako). Primary antibodies were applied at ambient temperature and antibody binding was visualized by incubation with Envision^™ Rabbit Link (Dako) followed by incubation with 3,3’-diaminobenzidine tetrahydrochloride (DAB). All the slides were subsequently counterstained with hematoxylin; they were dehydrated, cleared and mounted for the assessment. Antibodies used for staining are listed in Supplemental Table 1.