Immunofluorescence analysis of cell-cell junctions

HUVEC and BJ cells seeded on glass bottom in multiwell plate were fixed in p-formaldehyde at 4% v/v in PBS, (Lonza; Basilea, Swiss), permeabilized with Triton X-100 at 0.5% v/v in PBS (Lonza; Basilea, Swiss) and then blocked with goat serum at 20% v/v in PBS (Lonza; Basilea, Swiss). Incubation with the respective antibodies against VE-cadherin (mouse monoclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA), FAP1α (rabbit polyclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA) were performed for O/N at 4 °C. F-actin detection was evaluated by Phalloidin-FITC (5 μg/mL, Sigma-Aldrich; Saint Louis, MO, USA) for 30 min, at RT in the dark. The staining with conjugated secondary antibodies (1:500, antimouse and antirabbit), the nuclei with DAPI (1:1000) and the subsequent confocal microscope analysis and quantifications were performed as described in [62 (link),63 (link)]. In particular, the final images were generated using Adobe Photoshop CS4, version 11.0. Quantifications were performed from multichannel images obtained using a 63× objective using ImageJ, marking either the cell perimeter or the nucleus as the region of interest and calculating integrated densities per area from the appropriate channel. A minimum of 50 cells were analyzed for each data set; the obtained mean value has been used to compare experimental groups.