Mouse anti vinculin monoclonal antibody

Cell attachment of osteoblasts was evaluated by measuring the number of cells attached to the Ti microfiber scaffolds after 3 and 24 h of seeding. The numbers of viable cells were quantified using a WST-1-based colorimetric assay (WST-1, Roche Applied Science, Mannheim, Germany). A culture well was incubated at 37 °C for 1 h with 100 μL of WST-1 reagent. The amount of formazan product was measured using a multi-detection microplate reader (Synergy^TM HT, BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm. Osteoblasts on Ti microfibers were also morphometrically observed using confocal laser scanning microscopy. Cells were fixed in 10% formalin after culture for 3 and 24 h. The cells were stained with the fluorescent dye rhodamine phalloidin (actin filament, red color, Molecular Probes, Eugene, OR, USA). Expression of the cell adhesive protein vinculin was examined with double staining using a mouse anti-vinculin monoclonal antibody (Abcam, Cambridge, MA, USA), followed by FITC-labelled anti-mouse secondary antibody (Abcam).

Spreading behavior and cytoskeletal arrangement of osteoblasts seeded onto resin materials were examined using confocal laser scanning microscopy. At 24 h after seeding, cells were fixed in 10% formalin and stained using fluorescent dye rhodamine phalloidin (actin filament, red color; Molecular Probes, Eugene, OR, USA). To observe the intracellular expression and localization of vinculin, a focal adhesion protein, cells were additionally stained with mouse anti-vinculin monoclonal antibody (Abcam, Cambridge, MA, USA), followed by FITC-conjugated anti-mouse secondary antibody (Abcam). The area, perimeter, and Feret’s diameter, and the density of rhodamine- and vinculin-positive areas were quantified using an image analyzer (ImageJ, NIH, Bethesda, MD, USA).