Rabbit anti phospho histone h3 ser10

EdU staining was visualized with the Click-iT EdU Alexa Fluor 594 Imaging Kit per manufacturers instructions (Thermo Fisher). Labeling with additional antibodies was performed after EdU labeling. Sections were blocked with 2% BSA in PBS for 30 minutes prior to incubation in primary antibodies overnight at 4°C. Primary antibodies used were mouse anti-Pax7-s (IgG1, 1:5; DSHB), rabbit anti-activated-caspase-3 (1:400; BD Pharmingen), rabbit anti-phospho-histone H3 (Ser10, 1:400; EMD Millipore), and rabbit anti-phospho-S6 ribosomal protein (Ser235/236, 1:200; Cell Signaling). Sections labeled for anti-pS6 were initially post-fixed with 4% paraformaldehyde for 20 minutes, rinsed in PBS, incubated in PBS with 0.5% Triton-X for 20 minutes, rinsed in PBS, and boiled in 0.1M sodium citrate prior to incubation in blocking solution. Slides were incubated in appropriate secondary antibodies conjugated to Alexa Fluor 488 for 1 hour, and subsequently counter stained with DAPI (Roche) for 5 minutes before cover slipping.

Two fixation methods were used as previously described (Huggins et al., 2020 (link); Min et al., 2016 (link)) with minor modifications. Briefly, to observe the expression of fluorescently labeled whole worms, they were fixed with 4% paraformaldehyde at room temperature for 20 min and further fixed with 70% ethanol. The specimens were stained with DAPI (Thermo Scientific, Germany, 62248) to stain DNA, and observed on a Leica Thunder Imager Live Cell microscope with an HC PL APO 63x/1.47 Oil CORR TIRF objective and DAPI, GFP, and TXR filter sets. Images were acquired with a Leica DFC9000 GT deep-cooled sCMOS camera and Leica LAS X imaging software. To observe the mitotic germ cells and progression of spermatogenesis in males, worms were dissected and fixed with 100% methanol at −20 °C for 10 min, followed by 100% acetone fixation at −20 °C for 10 min. The specimens were further counter-stained with DAPI to stain DNA. The following primary and secondary antibodies were used: mouse monoclonal anti-α-tubulin (1:500; Sigma, T9026), rabbit anti-phospho-histone H3 (Ser10) (1:500; EMD Millipore, 06–570), Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500, Invitrogen, A32723), Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:500, Invitrogen, A32740).