Monoclonal anti vinculin

Confocal laser scanning microscopy (CLSM) and FE-SEM observation were used to investigate the morphology, cytoskeletal arrangement and focal adhesion development in adherent MSCs on the investigated samples at 4 and 24 hrs of culture. Cells were cultured on Ti disks at an initial seeding density of 1×10⁴ cells/well. The distribution of vinculin and organization of the actin filaments of the attached MSCs on the investigated samples was evaluated by CLSM (LSM700; Carl Zeiss, Oberkochen, Germany), which were identified following the double staining of actin (green fluorescence) and vinculin (red fluorescence) using diluted monoclonal anti-vinculin (Sigma–Aldrich, St. Louis, MO, USA), goat-anti-mouse IgG (Invitrogen, Carlsbad, CA, USA), and fluorescein isothiocyanate-labeled phalloidin (Sigma–Aldrich) according to a method described previously.9 (link) For the FE-SEM observation, MSCs spread on the investigated samples were fixed with 2% glutaraldehyde and 1% osmium tetroxide, then dehydrated using an ascending series of alcohols. After critical point drying and gold–palladium coating, the cell morphologies were observed using FE-SEM.

Snap frozen heart tissue and harvested cells were homogenized in RIPA buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris, pH7.4) in the presence of complete protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail 1 (Sigma) with the addiction of 1 mM N-Ethylmaleimide (NEM, Sigma) to prevent oxidation. All the procedures were performed at 4 °C. Protein concentration was determined by BCA assay (Pierce, Rockford, IL). Upon centrifugation, supernatant was diluted in Laemmli buffer 4X (Invitrogen) supplemented with 2.5% β-mercaptoethanol for reducing gels. Fifteen μg of tissue homogenate or 20 μg of cell homogenate were separated by 3–8% Tris-acetate (NuPage, Invitrogen) or 4–12% Bis-Tris (BioRad) and then transferred onto polyvinylidene difluoride (PVDF, BioRad) membranes. The following antibody were used: Monoclonal anti-Opa1 (1:1000 BD), rabbit polyclonal anti-GRP75 (1:1000 Santa Cruz Biotechnology), monoclonal anti-Vinculin (1:10,000 Sigma). Membranes were probed using isotype-matched secondary antibodies conjugated to horseradish peroxidase. Signal was detected with ECL (Amersham).