Sc 30044

hDPSCs cultured in 24-well plates were fixed with 4% PFA in PBS for 20 min at room temperature. After washing in PBS, samples were permeabilized with 0.1% Triton X-100 for 5 min and blocked with 1% BSA in PBS. Incubation with primary anti-OC antibody (sc-30044 Santa Cruz) was performed overnight at 4°C. Primary antibody was revealed using a FITC-conjugated anti-rabbit IgG secondary antibody. Nuclei were stained with Hoechst.
Frozen sections were stained by Hematoxylin and Eosin or permeabilized with 0.1% Triton X-100 for 15 min. Incubation with primary antibodies anti-OPN (ab8448 Abcam, Cambridge, UK), anti-OC (sc-30044 Santa Cruz), and anti-VEGF (ab39250 Abcam Cambridge, UK) was performed overnight at 4°C. FITC conjugated secondary antibody to rabbit IgG was used to reveal primary antibodies. Micrographs were taken with a microscope EVOS FL Cell Imaging System (Life Technologies).

At 14 days post PDL cell seeding, the cells were fixed with 4% formaldehyde for 10 min, followed by permeabilized within PBS containing 0.2% Triton X-100 and blocked in PBS containing 1% bovine serum albumin (BSA, Sigma) for 1 h. Subsequently, cells incubated overnight at 4 °C either with a polyclonal rabbit to collagen type I antibody (sc-28657, Santa Cruz, CA, USA) or a polyclonal rabbit to osteocalcin antibody (sc-30044, Santa Cruz) at a dilution of 1:75 in PBS containing 1% BSA. After washing with PBS, cells were incubated for 1 h at 37 °C with TR-conjugated-goat-anti-rabbit antibodies (sc-2780, Santa Cruz) (1:100) diluted in PBS containing 1% BSA. Prior to viewing, samples were mounted with Vectashield containing DAPI nuclear staining (Vector, Burlingame, CA, USA). Images were captured from each surface with an OLYMPUS BX51 fluorescence microscope. The optical density (OD) of the fluorescent staining was quantified from 3 independent experiments using Image J software.