Anti dmc1

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).

Adult testes were fixed in Bouin's solution overnight at 4°C, washed with PBS and 70% ethanol, embedded in paraffin, and sectioned at 5 µm thickness. Slides were matured overnight, de-waxed, rehydrated, and heated in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval. Sections were incubated in 3% hydrogen peroxide for 5 min and blocked in 2.5% normal horse serum (Vector Laboratories) for 80 minutes at room temperature. Later, slides were incubated overnight with anti-STRA8 (Abcam, 1∶500) or anti-DMC1 (Santa Cruz Biotechnology, 1∶50 dilution). The following day, slides were washed three times in PBS and incubated with anti-rabbit ImmPRESS peroxidase reagent (Vector Laboratories) for 30 minutes. The slides were later developed using a DAB substrate kit (Vector Laboratories) for 1 minute. The slides were counterstained with Mayer's hematoxylin for 5 minutes and washed in running water, dehydrated, and mounted with Permount (Fisher Scientific).

Tissue samples were fixed overnight in 4% paraformaldehyde followed by dehydration, embedding, and sectioning following standard protocols. Sections were subjected to H&E staining for routine analysis.
For immunofluorescence, sections were deparaffinized, rehydrated in a descending ethanol row, and rinsed with water. Heat-induced epitope retrieval and immunofluorescence were performed as described previously (Yao et al., 2020 (link)). The sections were blocked with 6% normal donkey serum for 1 h at room temperature, followed by incubation overnight with anti-SYCP3 (dilution: 1:25, catalog number: AF3750; R&D Systems, Minneapolis, MN, United States), anti-γH2AX (dilution: 1:400, catalog number: 2668445; Merck Millipore, Billerica, MA, United States), anti-DMC1 (dilution: 1:200, catalog number: sc-373862; Santa Cruz Biotechnology, CA, United States), and PNA antibodies (dilution: 1:400, catalog number: L21409; Life Technologies, Waltham, MA, United States) at 4°C in a humidity chamber. The sections were washed thrice with PBS-Tween, and incubated with highly cross-adsorbed secondary antibody conjugated with Alexa Fluor 488 or Alexa Fluor 555 (dilution: 1:400, Life Technologies) for 1 h at room temperature. After three washes, the nuclei were stained with Hoechst 33342. The images were captured by fluorescence microscope (Leica, Wetzler, Germany).