Envision plus reagent

Cells cultured on cover slides were fixed using 4% paraformaldehyde for 20 min at 4 °C and then washed three times with 10 mM PBS, pH 7.2. These cover slides were then incubated with anti-collagen I (1:500, ab6308, Abcam, Cambridge, UK), anti-osteopontin (1:1000 dilution, ab8448, Abcam, Cambridge, UK), and anti-osteonectin (1:500 dilution, AB 1858, Chemicon, Carlsbad, CA) antibodies for 24 h, followed by signal development using EnVision Plus reagent (Dako, Carpinteria, CA, USA), using diaminobenzidine as the chromogen and Mayer’s hematoxylin as the counterstain.
The relative staining intensity was scored arbitrarily on the light microscopy image as follows: no or weak staining (-), low intensity (1+), moderate intensity (2+), and strong intensity (3+).

The cells grown on coverslips were fixed using 4% paraformaldehyde for 20 min at 4°C and then washed with 10‐mM Tris‐HCl buffer. Then, they were incubated with the indicated primary antibodies: anti‐osteocalcin (predilution, AM 386; BioGenex), anti‐osteopontin (1:1000 dilution), and anti‐osteonectin (1:500 dilution, AB 1858; Chemicon) for 24 h, followed by development using EnVision Plus Reagent (Dako), diaminobenzidine as a chromogen, and Mayer's hematoxylin as a counterstain. Microscopic images were captured with a Nikon digital camera attached to a Nikon Optiphot‐2 microscope.