In Situ Hybridization for Collagen X and Osteocalcin

In situ hybridization was performed as described previously^{43 (link)}. Briefly, riboprobes for collagen X and for Osteocalcin⁴⁴ were generated by PCR using mouse growth plate cDNA as template and primers that contained an SP6 promoter. Single-stranded digoxigenin-labeled riboprobe for in situ hybridization was transcribed using a DIG RNA Labeling Kit (Roche Diagnostics) following the manufacturer’s protocol. Riboprobes were purified by Micro Bio-Spin Columns P-30 Tris RNase free (Bio-Rad). Paraffin-embedded sections of newborn bone/growth plate were hybridized to digoxigenin-labeled riboprobes (100 ng riboprobe per slide or 50 ng per section). Antigen retrieval was performed by proteinase K incubation (10 μg/ml, 37 °C for 30 min). 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 (1 h for Collagen X; overnight for Osteocalcin). Sections were counterstained with 10% eosin and visualized using Keyence BZ-X700 fluorescence microscope (Keyence Corp, Osaka, Japan) at ×10 magnification under bright field.