Immunohistochemical staining of cathepsin K and osteocalcin positive cells in hind paws

Left hind paw samples, from the ankle joint, collected at the time of sacrifice were fixed immediately in 10% neutral buffered formalin solution and then decalcified in 10% formic acid. Samples were next dehydrated and embedded in paraffin, serially sectioned at a thickness of 5 µm using a microtome, and mounted on microscope slides. Osteoclasts and osteoblasts immunolocalisation in subchondral bone tissue at the tibia/talus region was performed by staining with cathepsin K (osteoclast marker; mature osteoclast enzyme; Biorbyt, Cambridge, UK) and osteocalcin (osteoblast marker; indicator of osteoblastic activity; Abcam, Cambridge, UK) primary antibodies followed by EnVision+ (Dako, Glostrup, Denmark). Colour was developed in solution containing diaminobenzidine-tetrahydrochloride (Sigma, Sintra, Portugal), 0.5% H₂O₂ in phosphate-buffered saline buffer (pH 7.6). Slides were counterstained with haematoxylin and mounted. Immunohistochemical evaluation of rat joints was performed in a blinded fashion using a semiquantitative score of 0–3 (0: 0%–25% staining; 1: 25%–50% staining; 2: 50%–75% staining; 3: more than 75% staining) of the positively stained cells per total cellular count in the tissue section.^{9 21 25} Slides were analysed using a Leica DM2500 microscope (Leica Microsystems, Wetzlar, Germany).