Rabbit anti γh2ax antibody

Specimens were fixed with Bouin’s fixative and embedded in paraffin. One section showing the largest cut surface was made for each specimen, and stained with hematoxylin and eosin (H&E) or Periodic-Acid Schiff (PAS). For immunofluorescence staining, tissues were fixed with 4% paraformaldehyde in PBS overnight at 4°C. Cryoprotection was then performed with solutions of 10, 15, and 20% (w/v) sucrose in PBS for 1 hour each in succession. Tissues were cryo-embedded in OCT compound (Sakura Finetechnical) and cut into 7-μm-thick sections. The primary antibodies used were rat anti-Tra98 antibody (1:500, 2 μg/ml; Bioacademia), rabbit anti-Stra8 antibody (1:1,500, 667 ng/ml; Abcam), rabbit anti-Sycp1 antibody (1:200, 5 μg/ml; Novus Biologicals), and rabbit anti-γH2AX antibody (1:500, 100 ng/ml; Abcam). Tra98 is a pan-germ cell marker. Sra8 and γH2AX were used for detecting initial phase of meiosis. Sycp1 indicates pachytene spermatocytes. For the secondary antibodies, Alexa Fluor 555-conjugated goat anti-rat was used for the anti-Tra98 antibody, Alexa Fluor 488-conjugated goat anti-rabbit was used for the anti-Stra8 and anti-γH2AX antibodies, and Alexa Fluor 555-conjugated goat anti-rabbit was used for the anti-Sycp1 antibody. Nuclei were counterstained with Hoechst33342 dye.