Whole-mount in situ Hybridization of Mouse Embryos

Whole-mount in situ hybridization was performed as described previously^{21 (link)}. Embryos were dissected at E9.5 and fixed with 4% paraformaldehyde in PBS for durations of 1 h to overnight. After dehydration using a methanol series, embryos were rehydrated with 0.1% Tween 20 in PBS (PBST), bleached with 6% H₂O₂ in PBS, treated with Protease K, and then hybridized with digoxygenin (DIG)-labeled probes at 68ºC overnight. Embryos were then washed with 5 × and 2 × SSC/50% formamide and PBST. To detect DIG-labelled probes, embryos were incubated with anti-DIG antibodies after treatment with blocking reagent for 1 h, and the signal was developed with BM purple AP substrate (Roche, Basel, Switzerland).