Immunofluorescence was performed according to the method as described previously [8] (link). In brief, the testicular biopsies were obtained from the two patients with NOA. The testicular tissue was fixed overnight in 4% paraformaldehyde at 4 °C, and then embedded in warm paraffin (60 °C). The biopsies were sectioned at 5 μm thickness. The tissue sections were dewaxed in xylene, re-hydrated in a descending alcohol gradient, and heated in sodium citrate buffer (90–98 °C) for 15 min for antigen retrieval. After blocking with 5% BSA for 1 h at room temperature, the sections were incubated overnight with anti-SYCP3 (dilution: 1:25; catalogue number: AF3750, R&D Systems), anti-γH2AX (dilution: 1:300; catalogue number: 2668445, Millipore), anti-DMC1 (dilution: 1:100; catalogue number: sc-373862, Santa Cruz) and PNA (dilution: 1:400; catalogue number: L21409, Thermo Fisher Scientific) at 4 °C. The sections were washed thrice with PBS-T (Phosphate buffer saline-Tween), and incubated with highly cross-adsorbed secondary antibody conjugated with Alexa Fluor® 488 or Alexa Fluor® 594 (dilution: 1:400; Thermo Fisher Scientific) for 1 h at room temperature. The sections were washed three times with PBST and counterstained with 4’,6-diamidino-2-phenylindole (DAPI) to label the nuclei. The images were captured by fluorescence microscope (Leica).
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