Embryo Fixation and Staging Protocol

Dechorionated embryos were transferred to a 1.5 mL microtube and mixed in 362.5 μL PBT (0.3% Triton X-100 in PBS), 12.5 μL 10x PBS, and 125 μL 16% formaldehyde, methanol-free (w/v) (Thermo Fisher Scientific, USA). Embryos in the 4% formaldehyde fixing solution (w/v) were shaken for 15 min at 200 rpm using a mini rotator/shaker (Thermo Scientific). Fixing solution was discarded, 500 μL heptane (Sigma-Aldrich, USA) and 500 μL methanol (Thermo Fisher Scientific, USA) were added, and samples were vigorously shaken by hand/vortex for 2 min. heptane, methanol, and embryos in the interphase were removed and discarded. Samples were washed 3 times with methanol before resuspension in 1 mL PBT containing 1 μL Hoechst 33,342 (20 mM) (Thermo Fisher Scientific, USA). After a 10 min incubation at RT, 2 × 1 min and 1 × 10 min washes with 1 mL PTB were carried out to remove excess dye. Embryos were then staged using the ECLIPSE Ts2 microscope (Nikon) based on Foe et al.,¹⁰ (link) and reference images for nuclear cycle divisions from others.³⁶ (link)^,37 Embryos in PBT were kept on ice during staging. Finally, PBT was removed, TRIzol was added to pooled embryos, and samples were stored at −80°C until RNA was isolated using a standard TRIzol RNA isolation protocol.