Monoclonal antibody against vinculin

Osteoblast morphology, cytoskeletal arrangement, and focal adhesion of cells were observed using disk-based confocal scanning microscopy (DCSM; BX51, Olympus, Japan) and Meta-morph software (Olympus, Japan). Cells samples were examined after culturing for 4 and 24 hrs. Osteoblasts were cultured on various surfaces at an initial seeding density of 5000 cells/cm². To investigate the effects of nanotopographical features on focal adhesion and spread of osteoblasts, distribution of vinculin, and organization of actin filaments were investigated by DCSM. Focal adhesion contacts and cytoskeletons were identified by incubating samples with a monoclonal antibody against vinculin (Sigma-Aldrich, St. Louis, MO, USA) and fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma-Aldrich). An appropriate secondary goat-anti-mouse antibody was used to visualize vinculin staining (Invitrogen, Carlsbad, CA, USA). Samples were mounted on glass slides using anti-fade reagent and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI; Invitrogen), and examined by DCSM.

Cells were treated as described in (Majewski et al. 2011 (link)), and then stained with Alexa 488-conjugated phalloidin to visualize the actin cytoskeleton. Staining for adhesive structure markers was also performed using monoclonal antibody against vinculin (Sigma-Aldrich, USA) and polyclonal antibody against talin (Santa Cruz, USA) at a dilution of 1:40. For negative controls, primary antibody was omitted. Images were collected with the Leica TCS SP5 confocal laser scanning microscope equipped with a 63× HCX oil CS UV 1.4 oil-immersion objective.