Ab15087

The following antibodies were used: rabbit polyclonal antibody against TERB1 (1:1000)^{21 (link)}, TERB2 (this study, 1:2000), MAJIN (this study, 1:2000), SUN1 (this study, 1:1000), Speedy A (this study, 1:3000), phos-HistoneH2A.X (Ser139) (Millipore, 05-636-25UG, 1:2000), SYCP1 (Abcam, ab15087, 1:1000), SYCP3 (Abcam, ab15093, 1:2000), SOX9 (Millipore, ab5535, 1:200), MSH4 (Abcam, ab58666, 1:500), c-Myc (Santa Cruz, sc-789, 1:1000), TRF1 (1:1000)^{40 (link)}, mouse monoclonal antibodies against MLH1 (BD, 550838, 1:200), SYCP3 (Abcam, ab97672, 1:2000), TRF1 (Abcam, ab66223, 1:1000), c-Myc (Santa Cruz, sc-40, 1:1000), FLAG (Sigma, F3165, 1:200), actin (Sigma, A2228, 1:2000), DMC1 (Abcam, ab11054, 1:100), Lamin B1 (Proteintech, 66095-1-Ig, 1:500), CDK2 (Santa Cruz, sc-6248, 1:100). Secondary antibody for western blot: Goat anti-Mouse IgG/HRP (Abclonal, AS003, 1:2000), Goat anti-Rabbit IgG/HRP (Proteintech, SA00001, 1:2000). Goat anti-mouse/rabbit secondary antibody for IF: DyLight 488 (Thermo, 35502, 35553, 1:500), DyLight 550 (Thermo, 84540, 84541, 1:500), DyLight 633 (thermos, 35512, 35562, 1:500).

Spreading and immunolabeling of testicular samples were performed according to the standard protocol (47 ). Briefly, testes were dissected, rinsed in PBS, and decapsulated. The remaining tissues were transferred into a separation medium (hypo extraction buffer containing 30 mM Tris pH8.2, 50 mM sucrose, 17 mM citric acid, 5 mM EDTA, 2.5 mM DTT, and 1 mM PMSF) for 30 min. Spermatocytes were released from the tubules by finely mincing with a razor blade in 0.1 M of sucrose solution. Twenty microliters of the mixture was added onto a glass slide preloaded with 500 μl of 1% paraformaldehyde (pH 9.2) and spread evenly. Slides were incubated in a humidified chamber for 2 h. For immunostaining, slides were blocked in 1× ADB (1% goat serum, 3% BSA, 0.2% Triton X-100) at room temperature for 10 min, followed by incubation with primary antibodies at 4 °C overnight. On the following day, slides were washed and incubated with secondary antibodies for 1 h and finally mounted with DAPI (1:200). Antibodies against SYCP1 (Abcam, ab15087, 1:100), SYCP3 (Abcam, ab97672, 1:100), MLH1 (Abcam, ab92312, 1:50), γH2AX (Millipore, 05-636, 1:50), and RAD51 (Abcam, ab133534, 1:100) were used.

After deparaffinization and antigen retrieval, 5% bovine serum was used to block sections at RT for 1 h, and specific primary antibody SYCP1 (1:200, ab15087; Abcam), γH2AX (1:400, 05-636; Millipore), PLZF (1:100, AF2944; R&D), SOX9 (1:500, AB5535; Millipore), STRA8 (1:200, ab49405; Abcam), SYCP3 (1:200, ab15093; Abcam), PIWILI1 (1:200, A2150; Abclonal), F-ACTIN (1:200, Ab130935), DDX4 (1:200, Ab13840) was used to incubate with sections at RT or overnight at 4°C. After washing the sections three times, the slides were incubated with the corresponding secondary antibody, fluorescent dye–conjugated FITC (1:150, 115-095-003; Jackson) or TRITC (1:150, 115-025-003; Jackson) for 1 h at RT (avoiding the light). DAPI was used to stain the nucleus. All images were captured with confocal microscopy (Leica TCS SP8).