Hamamatsu color camera

For phalloidin staining to elicit F-actin organization, cells were fixed for 10 min using 4% paraformaldehyde followed by washing with PBS. Cells were then stained with Alexa Fluor® 594 phalloidin (300 units; Life Technologies, Carlsbad, CA) in PBS for 20 min at room temperature. Following staining, cells were washed with PBS and stained with DAPI for 4 min. Cells were then washed and mounted with Fluoromount G (Southern Biotech, Birmingham, AL). Fluorescence images were obtained using an Olympus FV3000 microscope (Olympus) equipped with a Hamamatsu color camera (Hamamatsu Photonics, Iwata City, Japan), using a 100x silicone oil immersion objective. Image analysis was performed with Olympus cellSens. Quantification of stress fiber anisotropy was performed using an Olympus cellSens Count and Measure Tool.

Human NHOst cells were grown to confluence in 4-well chamber slides (Sarstedt, Numbrecht, Germany), fixed for 10 min at room temperature with 4% paraformaldehyde (EMD Biosciences), then circled with an ImmEdge Hydrophobic Barrier Pen (Vector Laboratories). Next, cells were permeabilized for 10 min using 0.2% Triton-X (Sigma) in PBS. Non-specific binding was blocked with Dako Universal Blocking Buffer (Dako Products) for 1 h at room temperature. The slides were incubated overnight at 4 °C with either mouse anti-human osteocalcin (R&D Systems, 10 μg/ml), rabbit anti-human RUNX2 (Abcam, 1:500), mouse anti-human alpha-smooth muscle actin (1:100, Abcam), rabbit anti-human alkaline phosphatase (1:100, Abcam), rabbit anti-human osteopontin (1:500, Abcam), or goat anti-human IL-6 (40 μg/mL, R&D Systems), Next, slides were incubated for 1 h at room temperature with either donkey anti-mouse 594, chicken anti-rabbit 594, donkey anti-goat 488, or goat anti-rabbit 488 (1:1000, Biotium). Cells were stained with DAPI (0.2 ng/μl) then mounted with Fluoromount G (Southern Biotech). The cells without the primary antibody served as negative controls. Images were viewed using an Olympus FV 3000 fluorescent microscope (Olympus) equipped with Hamamatsu color camera (Hamamatsu Photonics).