Immunostaining Embryos for Diverse Markers

For immunostaining, embryos were fixed overnight at 4 °C with 4% formaldehyde (PFA) in phosphate-buffered saline (PBS) (pH 7.4), embedded in paraffin wax and serially sectioned at 8 μm. Immunostaining was performed either on whole mount embryos or on paraffin sections, as previously described (Burstyn-Cohen and Kalcheim, 2002 (link); Kahane and Kalcheim, 2020 (link)). Antibodies were diluted in PBS containing 5% fetal bovine serum (Biological Industries Israel, 04-007-1A) and 1% or 0.1% Triton X-100 (Sigma Aldrich Israel, X-100), respectively. Antibodies used were: rabbit anti-GFP (1:1000, Invitrogen, Thermo-Fisher Scientific, A-6455), mouse anti-GFP (1:100, Abcam, Ab38689), rabbit anti-RFP (1:1000, OriGene, AP09229PU-N), guinea pig anti-pSmad1/5/8 (1:300, a gift from E. Laufer), mouse anti-H3-pS10 (1:400, Abcam, Ab14955), rabbit anti-Laminin (1:100, Sigma Israel, L9393), rabbit anti-Sox9 (1:150, Millipore, AB5535), rabbit anti-SNAI2 (1:500; Cell Signaling Technologies, 9585), mouse anti-CD57 (anti-HNK1, 1:500, BD Biosciences, 559048), rabbit anti-BarHL1 (1:300, Sigma Israel, HPA004809), mouse anti-Pax7 (1:10, DHSB, pax7), mouse anti-Hb9 (1:200, DHSB, 81.5C10). Cell nuclei were visualized with 125 ng/ml Hoechst 33,258 (Sigma Israel, 14530) diluted in PBS.