The procedure for in situ hybridization was described previously 20 (link). Briefly, RT-PCR was performed using mRNA from tooth germ of miniature pigs. The correct size bands were extracted from agarose gels and DNA sequencing was performed. The RNA probe was synthesized by in vitro transcription according to the protocol of DIG RNA labeling Mix (Roche). For the staining procedure, after serial rehydration, the slides were treated with proteinase K (1 μg/ml in PBS) for 30 min at 37°C. After being re-fixed with 4% paraformaldehyde, the sections were dehydrated in series of ethanol (25, 50, 75 and 100%). After being dried for 1 h, the specimens were hybridized with probe at 70°C overnight. After being washed for hours, the sections were incubated with alkaline phosphatase conjugated anti-digoxigenin Fab (Roche) overnight. Signals were detected with NBT/BCIP substrates (Promega). Primers used for RT-PCR were list as follows:
WNT5a (forward: 5'-ctggcaggactttctcaagg-3';
reverse: 5'-cgcgctgtcatacttctcct-3');
β-Catenin (forward: 5'-ggtccatcagctttccaaaa-3';
reverse: 5'-ctgaacaagggtcccaagaa-3');
Axin2 (forward: 5'-gagggagaaatgcgtggata-3';
reverse: 5'-tgggtgagagtttgcacttg-3').
