Fluoview fv1000 confocal

Twenty-four h after transfection the cells were fixed with 3% paraformaldehyde in phosphate buffered saline (PBS) for 15 minutes at room temperature. Cells were then washed with PBS three times, fixed with 3% paraformaldehyde (PFA), and the coverslips were mounted onto microscope slides. Samples were visualized using a Fluoview FV1000 confocal laser-scanning microscope (Olympus).

Cells cultured in the FEPM devices were fixed with 4% paraformaldehyde (PFA, Nacalai Tesque, Kyoto, Japan) for 20 min, washed with PBS, and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, USA) for 15 min. The cells were then incubated with blocking buffer containing 1% bovine serum albumin (BSA, Sigma-Aldrich) for 30 min at room temperature and incubated with antibodies directed against vascular endothelial (VE)-cadherin (Abcam, ab33168, dilution 1:200, Cambridge, UK) or Claudin (Abcam, ab15098, dilution 1:200) overnight at 4 °C, followed by PBS washes. Subsequently, Alexa 568-conjugated secondary antibody (Abcam, ab175471, dilution 1:500) was introduced into the channels and incubated for 1 h at room temperature. The cells were co-stained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen, D1306, Waltham, USA). Cross-sectional images were obtained at 2 µm intervals in the vertical direction using a confocal microscopy (FluoView FV1000 confocal, Olympus, Tokyo, Japan) with the 10× objective.