Immunofluorescence Analysis of Osteogenic Markers

Immunofluorescence analyses were performed as previously reported [30 (link)]. Briefly, cells were fixed and processed according to the manufacturer’s protocols. Primary antibodies for RUNX2 and osteocalcin (sc74495, Santa Cruz, Dallas, Texas, USA) were diluted according to the manufacturer’s instruction in antibody dilution buffer and incubated overnight at 4°C. Slides were then incubated with the secondary antibodies goat mouse fluorescein conjugated (cat. Ap124f, Millipore, Burlington, Massachusetts, USA). Nuclear staining was performed by ProLong™ Gold Antifade Mountant with DAPI. The staining was analyzed using a Leica (Wetzlar, Germany) TCS SP5 AOBS microscope. To express data in a semiquantitative way, six different fields were analyzed for each sample, in three independent experiments with about 80–100 total cells.