In situ hybridization
The pCRII-TOPO TA vector containing a region of the 3′ UTR of Snail was used as a template to generate digoxigenin-labeled sense and antisense riboprobes (Roche). The respective probes were obtained by XhoI and BamH1 digestions. In situ hybridizations were performed on 10-μm thick sections of E17.5 mouse embryos. The sections were fixed with 4% PFA for 10 min at room temperature, prehybridized at room temperature for 4.5 h, hybridized with the probe (2 μg/ml) at 55 °C for 12–14 h, blocked with 10% NGS, and treated with anti-dig Fab-AP antibody (Roche #1093274) at a 1:2,500 dilution for 3 h. The sections were incubated with NBT and BCIP until adequate signal was detected.