Vectashield antifade mounting medium with dap

The meiotic chromosome spreads were prepared as described in a previous study [68 (link)], using a solution mixture of 1% paraformaldehyde (PFA) and 0.15% Triton X-100, with pH adjusted to 9.2 with 1 N HCl or 1 N NaOH, as the fixative. The slides were incubated with primary antibodies at 4 °C for 2 h and with secondary antibodies at room temperature for 1 h. The chromosome spreads were mounted in VECTASHIELD Antifade Mounting Medium with DAP (Vector Laboratories, Burlingame, CA, USA).

The TUNEL assay was performed using the TUNEL Assay Kit-BrdU-Red (ab66110; Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions but with some modifications according to experimental conditions. Briefly, after treating with primary anti-Sycp3 antibody for 3 h at 4 °C, the spermatocytes were labeled with rTdT reaction mix for 1 h at 37 °C. After washing in PBS, the sections were incubated with anti-BrdU antibody for 30 min at room temperature and washed in PBS. After washing, the slides were incubated with Alexa Fluor 488 Goat Anti-Rabbit Antibody (A11034; ThermoFisher Scientific, Rockford, IL, USA) for 30 min at room temperature. Then, the slides were mounted with VECTASHIELD Antifade Mounting Medium with DAP (Vector Laboratories, Burlingame, CA, USA). The images were visualized by confocal microscopy (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany) and analyzed using the Leica LAS AF Software lite 3.2.0.