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Fluorescent staining of osteoclasts to assess F-actin ring formation

PP Paula Pennanen
RK Roope A. Kallionpää
SP Sirkku Peltonen
LN Liisa Nissinen
VK Veli-Matti Kähäri
EH Eetu Heervä
JP Juha Peltonen
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The effects of the different inhibitor treatments on osteoclast differentiation on bovine bone slices were studied by fluorescent staining of nuclei and F-actin rings. First, osteoclasts were fixed with 4% paraformaldehyde, permeabilized with 0.01% Tween-20 in PBS on ice for 5 min, and non-specific binding was blocked with 1% BSA in PBS for 30 min. Cells were stained with STAR635 phalloidin (Abberior GmbH, Gottingen, Germany; catalogue number 2-0205-002-5) in 1:100 dilution to visualize F-actin and with Hoechst 33342 at a 1:10000 dilution in 1% BSA in PBS to visualize the nuclei, both for 1 h at room temperature. Glass coverslips were embedded with 30% glycerol in PBS and imaged with Carl Zeiss Axioimager microscope and Carl Zeiss Zen 2012 (blue edition) software.

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