Mouse monoclonal anti vinculin primary antibody

Cells were fixed with 2% paraformaldehyde (Santa Cruz Biotechnologies, Dallas, TX, United States) for 15 min at room temperature, then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 10 min and blocked with 4% BSA (Sigma-Aldrich). They were incubated with mouse monoclonal anti-vinculin primary antibody (1/150, Sigma-Aldrich) or mouse monoclonal anti-desmin antibody (1/100, DAKO, D33) for 1 h at room temperature. After three washes with PBS, cells were incubated 45 min with isotype-specific anti-mouse secondary antibody labeled with Alexa-488 (Molecular Probes) or anti-rabbit secondary antibody labeled with Alexa-568 (Molecular Probes). DNA was stained with Hoechst 33258 (1 μg/ml, Sigma-Aldrich) during secondary antibody incubation. Finally, cells were washed in PBS and mounted in Fluoromount medium (Interchim). Images were acquired by confocal microscopy (LSM700 Zeiss) at the imaging facility of the Functional and Adaptive Biology (BFA) unit.