Ab22552

To localize, within the osteotomies, cells that had initiated differentiation down an osteogenic lineage, immunostaining was performed using standard procedures [18 (link)]. In brief, following deparaffinization, endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 5 min, and then washed in PBS. Slides were blocked with 5% goat serum (Vector S-1000) for 1 h at room temperature. The appropriate primary antibody was added and incubated overnight at 4 °C, then washed in PBS. The primary antibodies used in this study were Osterix (1:1200; ab22552, Abcam, Cambridge, MA, USA) and Cathepsin K (1:200; ab19027, Abcam, Cambridge, MA, USA). Samples were incubated with appropriate biotinylated secondary antibodies (Vector BA-x) for 30 min, then washed in PBS. An avidin/biotinylated enzyme complex (Kit ABC Peroxidase Standard Vectastain PK-4000, Vectorlabs, Burlingame, CA, USA) was added and incubated for 30 min, and a 3,3′-diaminobenzidine (DAB) substrate kit (Kit Vector Peroxidase substrate DAB SK-4100, Vectorlabs, Burlingame, CA, USA) was used to develop the color reaction. Phalloidin immunostaining was performed using Palloidin Control, DyLight 488 conjugate (1:300; PI21833, Invitrogen, Grand Island, NY, USA).

Cell extracts were prepared using Mammalian Protein Extraction Reagent (M-PER; Thermo Fisher Scientific) with complete protease inhibitor cocktail (Thermo Fisher Scientific) according to the manufacturer's instructions. Western blotting was performed by standard methods with anti-phospho p42/p44 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling Technology Inc., Danvers, MA), anti-total ERK1/2, anti-Runx2 (1:1000, ab76956, Abcam, Cambridge, MA), anti-Osterix (SP7) (1:1000, ab22552, Abcam), and anti-β-actin (1:1000, A5441, Sigma-Aldrich) antibodies.