Immunohistochemical Analysis of PRM1 and PRM2

Bouin’s fixed testicular tissue was processed in paraffin wax. Immunohistochemistry (IHC) against PRM1 was performed as described previously64 (link) using the Autostainer 480 S (Medac, Hamburg, Germany). For IHC of PRM2, sections were treated for 10 min at RT with decondensing-mix containing 25 mM dithiothreitol, 0.2% Triton X-100 and 200 IU heparin/ml in PBS to enhance antigen accessibility for the primary antibody69 (link). Subsequently, sections were washed in 0.02 M PBS (pH 7.4), boiled for 20 min in sodium citrate buffer, treated for 20 min with 3% H₂O₂ in methanol and blocked for 20 min with 5% bovine serum albumin (BSA) in PBS. Incubation with the primary antibody was performed overnight at 4 °C, with biotinylated goat-anti-mouse secondary antibody (DAKO) and the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA) for 1 h at RT each. Immunoreaction was visualized using AEC (DAKO). Finally, sections were counterstained with hematoxylin and covered with glycerin gelatin.