In situ Hybridization of Mouse Growth Plates

In situ hybridization was performed as described previously39 (link). Briefly, riboprobes were generated by PCR using mouse growth plates cDNA as template and with primers that contained SP6 promoter (primer sequence for probe provided upon request). Single stranded digoxigenin-labelled riboprobes for in situ hybridization were transcribed using the DIG RNA Labelling Kit (Roche Diagnostics) following the manufacturer’s protocol. Riboprobes were purified by Micro Bio-Spin Columns P-30 Tris RNase free (Bio-Rad), followed by alkaline hydrolysis for 30 min. Paraffin-embedded sections of epiphyseal cartilage from 1-week-old mice were hybridized to digoxigenin-labelled riboprobes. For detection, tissue sections were incubated with anti-digoxigenin alkaline phosphatase Fab fragments (Roche) for 2 h at room temperature and treated with NBT/BCIP (Sigma) in the dark until a colorimetric change was detected. Sections were counter-stained with 10% eosin and visualized using a ScanScope CS digital scanner (Aperio Technologies, Inc) under bright-field microscopy.