Alexa fluor 488 and 568

Embryos were fixed at the desired stages for 1–3 h using MAD fixative (2 parts methanol, Thermo Fisher; 2 parts acetone, Thermo Fisher; 1 part DMSO, Sigma). After fixation, embryos were dehydrated in methanol and stored at −20°C. Embryos were then processed for immunofluorescence as previously described^{25 (link)}. Briefly, embryos were gradually rehydrated in 0.5X SSC (1X SSC: 150 mM NaCl, 15 mM Na citrate, pH 7.0), then bleached with 2% H₂O₂ in 0.5X SSC with 5% formamide for 2 h under light. Embryos were washed with PBT (1X PBS, 0.1% Triton X-100, 2 mg/mL bovine serum albumin). Embryos were blocked in PBT supplemented with 10% goat serum and 5% DMSO for 1–3 h and incubated overnight at 4°C in PBT supplemented with 10% goat serum and primary antibodies. We used mouse anti-beta-tubulin (E7, Developmental Studies Hybridoma Bank, 1:300 dilution) and rabbit anti-histone H3 (Abcam, 1:500 dilution). Embryos were then washed 4 × 2 h in PBT and incubated overnight at 4°C in PBT supplemented with goat anti-mouse and goat anti-rabbit secondary antibodies coupled to Alexa Fluor 488 and 568 (Invitrogen). Embryos were then washed 4 × 2 h in PBT and gradually dehydrated in methanol. Finally, embryos were cleared in Murray’s clearing medium (2 parts benzyl benzoate, Sigma; 1 part benzyl alcohol, Sigma). Embryos were placed in a reusable chamber (Thermo Fisher) for confocal microscopy.