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.
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.