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).
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).
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