Mouse monoclonal antibodies against vinculin

After culturing for 3 d, the cells were incubated in PBS-0.05% Triton X-100 (PBST) containing 5% normal goat serum (Thermo Fisher Scientific) for 30 min to block the non-specific antibody-binding sites. The cells were incubated with mouse monoclonal antibodies against vinculin (Sigma-Aldrich) at 4 °C for 12 h and then Alexa Fluor 546-conjugated anti mouse IgG (Molecular Probes, Invitrogen, Thermo Fisher Scientific), Alexa Fluor 488-conjugated phalloidin (Molecular Probes, Invitrogen), and Hoechst33342 (Nacalai Tesque). Finally, the cells were washed with PBST and mounted in the Prolong Diamond Antifade reagent (Thermo Fisher Scientific). Fluorescent images were obtained using a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan).

Cells were fixed in 4% paraformaldehyde and incubated in PBS-0.05% Triton X-100 (PBST) containing 1% normal goat serum (NGS; Invitrogen) for 30 minutes to block non-specific antibody binding sites. The cells were then incubated with mouse monoclonal antibodies against vinculin (Sigma-Aldrich) at 4 °C for 12 h. This step was followed by incubation with Alexa Fluor 546 conjugated anti mouse IgG (Molecular Probes, Invitrogen), Hoechst (invitrogen), and Alexa Fluor 488 conjugated phalloidin (Molecular Probes, Invitrogen). Finally, the cells were washed with PBST and mounted using the Prolong Gold antifade reagent (Molecular Probes, Invitrogen). Fluorescent images were observed using a fluorescence microscope, and processed using the Adobe Photoshop 10.0 software.
To identify the cancer cells, the following antibodies were applied as cancer cell markers; mouse anti MelanA (Santa Cruz) for B16F10, mouse anti CathepsinD (Abcam) for MDA-MB-231, and mouse anti PSA (Abcam) for MDA-PCa-2b. Intercellular adhesion was visualized using the following antibodies: rabbit-anti Connexin 43 (Cell Signaling) and rabbit anti-Cadherin 11 (Cell Signaling).