After culturing the cells were fixed with 4% PFA in PBS for 10 min at RT. The actin cytoskeleton was stained with Alexa 488-conjugated phalloidin (200 U/ml stock diluted 1:100 in PBS; Invitrogen Europe, Paisley, UK) for 20 min at + 37 °C. Nuclei were stained with Hoechst 33258 (1 mg/ml stock diluted 1:800 in PBS; Sigma-Aldrich) for 10 min at room temperature (RT). Staining for osteoclast-specific enzyme TRACP was carried out with a commercial acid phosphatase leukocyte kit (Sigma-Aldrich) for 20 min at + 37 °C. The samples were mounted in 70% glycerol-PBS and viewed in a Zeiss Axio Scope.A1 fluorescence microscope (Oberkochen, Germany) and EC Plan Neofluar 20 × objective. Multinuclear cells with three or more nuclei were counted from each bone slice from five randomly chosen microscope fields, bone slice n = 4–6. The number of nuclei per cell were counted from 5 multinuclear cells from 5 randomly chosen areas. Images were taken with Nikon Eclipse E600 fluorescence microscope using Plan 20 ×/0.5 objective (Tokyo, Japan), QImaging MicroPublisher 5.0 RTV camera and QCapture 2.90.1 software (QImaging, Surrey, Canada). Confocal images were taken with Leica TCS SP8 confocal with a DMI8 microscope using LAS X 3.5.2 acquisition software. The objective used was an HC PL APO CS2 20 ×/0.75 DRY. Samples were imaged with 488 nm and 405 nm solid-state lasers; the pinhole was set to Airy 1 and scan speed to 600 Hz.
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