Immunohistochemical assessment was performed at 2 weeks and 3 weeks after the fracture (n=5 in each group). We assessed Ki67 expression of chondrocyte within bony callus because it is one of the best-known proliferation markers.11 12 (link) We also evaluated cathepsin K expression as an osteoclast marker.13 14 (link) The sections were incubated overnight at 4℃ with anti-Ki67 antibody (1:50 dilution, NB500-170, Novus Biologicals, Centennial, Colorado, USA) or anticathepsin K antibody (1:50 dilution, ab19027, Abcam, Cambridge, Massachusetts, USA) and subsequently treated with peroxidase-labeled antimouse immunoglobulin (Histofine Simple Stain MAX PO (R), Nichirei Bioscience, Tokyo, Japan) at room temperature for 60 min. The signal was developed as a brown reaction product using the peroxidase substrate 3,3’-diaminobenzidine (Histofine Simple Stain 3,3′-Diaminobenzidine (DAB) Solution, Nichirei Bioscience). The sections were counterstained with hematoxylin and examined with a BZ-X700 confocal microscope (Keyence Corporation, Osaka, Japan). Immunopositive cells were counted in four random fields under a high-power field.
All morphometric studies of immunofluorescence and immunohistochemical staining were performed by two blinded orthopedic surgeons.
All morphometric studies of immunofluorescence and immunohistochemical staining were performed by two blinded orthopedic surgeons.
