Mca757g

Tartrate-resistant acid phosphatase (TRAP) staining of formalin-fixed osteoclasts used naphthol AS-BI phosphate as a substrate, reacting the product with fast violet B salt at 37˚C for 3 h. TRAP-positive multinucleated cells containing 3 ≥ nuclei were considered osteoclasts. Vitronectin receptor (VNR, CD51/61, αVβ3-integrin) expression was determined by immunohistochemistry using a murine monoclonal antibody targeting CD51/61 (MCA757G; AbD Serotec, Oxford, UK) and visualised with DAB. Immunofluorescent staining of F-actin filaments was performed on formalin-fixed cells for 30 min using TRITC-conjugated phalloidin (Sigma-Aldrich, Poole, UK), mounted with fluoroshield containing DAPI (Sigma)and visualised at 450–480 nm. Image acquisition was performed using a Zeiss AxioImager MI microscope, AxioCam HRC camera and AxioVision software. Quantification was performed using ImageJ software (National Institutes of Health, Bethesda, USA) to count the average number of osteoclasts per field of view. Fields of view were randomly selected using pre-defined and consistent locations within each well, counting 4 fields of view at 4 × magnification per well with triplicate wells per experimental condition. To avoid observer bias, counting was performed while blinded to the image identity.