Immunocytochemical analysis was performed using an ImmPRESS Universal Reagent kit (Vector Laboratories, Inc.). hOCs or hOBs (1×106/cm2 and 1×104/cm2 cells, respectively) were seeded in 24-well plates, fixed in cold 100% methanol at room temperature for 10 min and permeabilized with 0.2% (v/v) Triton X-100 in TBS (1X). Then, the cells were treated in 0.3% H2O2 in TBS (1X) for 10 min at room temperature, and subsequently incubated with ready-to-use (2.5%) normal horse serum blocking solution (ImmPRESS Universal Reagent kit) for 15 min at room temperature.

After the incubation in blocking serum, cells were incubated at 4°C overnight following addition of the following rabbit anti-human polyclonal primary antibodies: Runx2 (1:200); COL1a1 (1:100); NFATc1 (1:300); cathepsin K (1:200); and OPN (1:200). After rinsing in 1X TBS, the cells were incubated for 30 min at room temperature with ImmPRESS reagent and then stained with substrate/chromogen mix (ImmPACT™ DAB). After washing, the cells were mounted in glycerol/PBS (9:1), counterstained with hematoxylin and observed with a Nikon Eclipse 50i optical microscope (magnification, ×20; Nikon Corporation).

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