Mouse anti human osteocalcin

Four populations of hMDSCs and hBMMSCs were subjected to pellet culture before and after lenti-BMP2 transduction using a protocol previously described [9 (link)]. Four to 8 replicate pellets were prepared for each population and cultured in osteogenic medium utilizing the same conditions. At 3 and 4 weeks after initiating the osteogenic cultures, the pellets were scanned with a microCT (Viva CT 40,Scanco Medical, Switzerland) to detect mineralization using 21voxel size and medium resolution. The mineralized pellet volumes were evaluated using the following parameters: Gauss Sigma 0.8, Gauss support 1.0, and threshold 122. After 4 weeks, the cell pellets were fixed in 4% neutral buffered formaldehyde for 2 hrs at RT, embedded in NEG 50 freezing medium, snap frozen in liquid nitrogen, and stored at -80°C until cryosectioned at 8 μm. Von Kossa staining was performed using an online protocol (IHC world) to verify the calcification of the pellets. Osteocalcin immunohistochemistry using mouse anti-human osteocalcin (R &D system, USA) primary antibody was also performed as previously reported [9 (link)]. DAB color reaction was used to reveal osteogenic differentiation of the cells as shown in brown. Pellet culture experiments were repeated three times for all cell populations.

Cells were cultured on 8 chamber slides at a density of 1 × 10⁴ cells per well. After 21 days of culture in osteoblast differentiation cocktail, cells were fixed with a 95% ethanol and 5% acetic acid for 10 minutes at −20°C. After washing with PBS, cells were permeabilized with 0.1% (v/v) Triton X-100 (Sigma) for 45 minutes and washed again with PBS. Nonspecific binding was blocked by incubating in 1:20 horse serum (Sigma) for 1 hour. Cells were then incubated with mouse anti-human osteocalcin (R&D Systems, Minneapolis MN) at 4°C overnight. After washing with PBS, cells were incubated with secondary antibody (donkey Alexa-Fluor-488) (Invitrogen, Carlsbad CA) for 1 hour at room temperature. Nuclear counterstaining was performed with mounting medium containing DAPI (Vector, Burlingame CA).