Non-Radioactive In Situ Hybridization Protocol

The procedure for non-radioactive in situ hybridization has been described previously (Wang et al., 2017 (link)). Briefly, RT-PCR was performed using mRNA from tooth germ of WZSPs. The correct size bands were extracted from agarose gels and DNA sequencing was performed. The primers used for pig Pitx2, Msx2, Left, Dlx2 were listed in Table S1. The RNA probe was made by labeling with digoxigenin-UTP by in vitro transcription with T7 RNA polymerase according to the protocol of DIG RNA labeling Mix (Roche). For the staining procedure, mandible samples were rinsed in RNAse-free PBS and fixed in 4% paraformaldehyde in PBS (pH 7.5). The fixed tissues were decalcified, embedded in paraffin and cut into slices (6 μm). After the rehydration, the slides were treated with proteinase K (1 μg ml⁻¹ in PBS) for 30 min at 37°C, and then re-fixed with 4% paraformaldehyde in PBS then rinsed with PBS. The specimens were then dehydrated with series of ethanol, before leaving the slides to air dry for 1 h. The specimens were hybridized in hybridization buffer at 70°C overnight. After washing for 3–4 h, specimens were incubated with alkaline phosphatase conjugated anti-digoxigenin Fab (Roche) overnight. Positive signals were detected by incubating the specimens with NBT/BCIP substrates (Promega).