Mouse anti runx2

After deparaffinization and rehydration, the sections were treated with pepsin (Zhongshan, Beijing, China) for 10 min. For immunohistochemical staining, endogenous peroxidase was blocked with 3% hydrogen peroxide followed by blocking with goat serum for 30 minutes. The sections were incubated with rabbit anti-β-catenin (Cell Signaling Technology, Beverly, MA, USA) and mouse anti-Runx2 (Abcam, Cambridge, UK) primary antibodies at 4°C overnight. The sections were washed and stained using the SP kit (Maixin-bio, Fuzhou, China) according to the manufacturer's instructions. Then stainings were visualized by diaminobenzidine solution (Maixin-bio, Fuzhou, China). For double immunofluorescence staining, the sections were incubated with primary antibodies mixtures of rabbit anti-β-catenin and mouse anti-Runx2 antibodies at 4°C overnight, followed by incubation with a mixture of goat anti-rabbit-Cy3 and goat anti-mouse-Dylight 488 antibodies for 1 h at room temperature. Sections were counterstained with DAPI to reveal nuclei. Photos were obtained by a fluorescent microscope with a camera (Leica, Wetzlar, Germany). Excitation and emission wavelengths were 405 and 425–460 nm (blue) for DAPI, 473 and 485–545 nm (green) for DyLight 488, 559 and 575–620 nm (red) for Cy3. Negative controls were obtained by substitution of primary antibodies with phosphate-buffered saline (PBS).