Ab19027

Paraffin-embedded, 3-μm tissue sections were mounted onto SuperFrost slides, deparaffinised in xylene and ethanol of graded concentrations. For antigen retrieval, the slides were treated in a microwave oven in a solution of TRS (Target Retrieval Solution, High pH, Dako, Denmark) for 30 min (2 × 6 min 360W, 2 × 5 180W, 2 × 4 min 90 W). After cooling down at room temperature, they were transferred to 0.3% hydrogen peroxide in methanol, for 30 min, to block endogenous peroxidase activities. Sections were rinsed with Tris-buffered saline (TBS, Dako, Denmark) and incubated with rabbit primary antibodies against: cathepsin K (Abcam, UK; ab 19027, dilution 1 : 200), and with mouse monoclonal antibody against VEGF (Dako, Denmark, clone VG1, dilution 1 : 300), CD34 (Dako, Denmark, clone QBend 10, dilution 1 : 50). Immunoreactive proteins were visualized using adequate EnVision-HRP kit (Dako, Carpinteria, CA, USA) according to the instructions of the manufacturer. Visualisation was performed by incubation of the sections in a solution of 3,3’-diaminobenzidine (Dako, Denmark). After washing, the sections were counterstained with Mayer’s haematoxylin and mounted.
For each antibody and for each sample, a negative control was processed. Negative controls were carried out by incubation in the absence of the primary antibody and always yielded negative results.

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.

Tissue sections were deparaffinized and rehydrated, then endogenous peroxidase was blocked with 3% H₂O₂/PBS solution. After blocking with 2% goat serum, sections were incubated with an antibody against Cathepsin K (ab19027, Abcam) overnight at 4 °C. After washing with PBS, sections were incubated with biotinylated goat secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 60 min at room temperature followed by treatment with Vectastain elite ABC kit (Vector Laboratories). Sections were colored using ImmPACT DAB (Vector Laboratories) and counterstained with hematoxylin.