Alexa 488 568 conjugated secondary antibodies

To obtain meiotic pachytene oocytes, embryonic ovaries of 17.5 d female embryos were isolated (Susiarjo et al., 2009 (link)). Ovaries were then incubated in a drop of hypotonic buffer (17 mM trisodium citrate-dihydrate, 50 mM sucrose, 5 mM EDTA, 0.5 mM DTT, and 30 mM Tris-HCl, pH 8.2; Sigma) for 25 min. Ovaries were disintegrated with 21 G needles to release cells in a sucrose drop (100 mM; Sigma). Cells were then fixed with 2% PFA containing 0.2% Triton X-100 overnight at RT in humidified box. Slides were air dried slowly. After air drying, pachytene oocytes were permeabilized with 0.1% Triton X-100 and were incubated with primary antibodies for 1.5 h at RT. Immunofluorescent staining was performed using rabbit anti-SYCP1 (Abcam, ab15090) and mouse anti-MLH1 (BD551092) primary antibodies. Appropriate Alexa 488/568 conjugated secondary antibodies (Invitrogen) were used for visualization and 1 µg/ml DAPI was applied for DNA counterstaining. Microscopy slides were prepared with Vectashield mounting medium. Imaging of spreads was performed on a Zeiss LSM780 confocal microscope equipped with a 40×/1.4 EC plan-Apochromat oil DIC objective lens using Zen Black software. For a more comprehensive protocol, see Silva et al. (2018) (link).

Nuclear extracts for Western blotting were prepared by resuspending cell pellets in hypotonic lysis buffer (10 mM Tris-HCl pH 7.5, 1.5 mM MgCl₂, 5 mM KCl, protease and phosphatase inhibitors), followed by nuclear extraction buffer (50 mM Tris-HCl pH 7.5, 0.5 M NaCl, 2 mM EDTA, 10% sucrose, 10% glycerol, protease and phosphatase inhibitors). Whole cell extracts were prepared by resuspending cell pellets in 1xSDS protein lysis buffer (67.5 mM Tris pH 6.8, 25 mM NaCl, 0.5 mM EDTA, 12.5% Glycerol, 0.25% SDS, DTT). The following primary antibodies were used: OGG1 (Abcam), APE1 (Santa Cruz Biotechnology), EXO1, RNase H2A (Bethyl), DNA2 (Thermo Fisher Scientific) and beta-actin (Cell Signaling). The following secondary antibodies were used: horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and Alexa488/568-conjugated secondary antibodies (Invitrogen). Antibody dilutions and catalogue numbers are provided in Supplementary Table 3. Uncropped gels are shown in Supplementary Figs. 4, 5.