In situ Hybridization of FAM20A in Mouse Mandible

For in situ hybridization (ISH) analyses, diethylpyrocarbonate-treated solutions were used for processing the mouse mandibles and for the hybridization experiments to ensure RNase-free conditions. We generated a 681-bp RNA probe that was complementary to the mouse FAM20A mRNA, using the same method as we previously described.^{11 (link), 24 (link)} Briefly, a primer set with 5′-ACAATTCAACCTTACCTCCTTGG-3′ (in exon 2 of the mouse Fam20A gene) for forward and 5′-CTTTTCCTGACAGCGAGTAGG-3′ (in exon 8) for reverse was used to generate a cDNA fragment using PCR, with total RNA extracted from the molars of 16.5-day-old mice as the template. Next, the PCR products were cloned into the pCRII-TOPO vector (Promega, Madison, WI, USA) and were transformed into competent Escherichia coli. The plasmid DNA was isolated from E. coli and sequenced. Digoxigenin-labelled single-stranded RNA probes were synthesized and labelled with digoxigenin using an RNA labelling kit (Roche, Indianapolis, IN, USA), and the DIG-labelled RNA probes were detected by enzyme-linked immunoassay with a specific anti-DIG-AP antibody conjugate, as we previously described.^{11 (link), 24 (link)} These RNA probes were used to detect FAM20A mRNA in the paraffin sections containing tissues from the mouse mandible.