Ve cadherin cd144

For confocal imaging, the cells were grown to 100% confluence on collagen-coated 8-chamber glass slides (BD Biosciences. Cat, no: 354108). Cells were fixed for immunocytochemical staining using 4% PFA at room temperature for 10 min. Excess PFA was removed with several washes using PBS. Primary antibody against VE-cadherin/CD144 (Santa Cruz Biotechnology Inc, sc-6458) was prepared in PBS containing 1% donkey serum and used at 1:200 dilution. Primary antibody to zonula occludin-1 (ZO-1; Invitrogen, 339100) was prepared in PBS containing 1% goat serum and used at 1:500 dilution. Primary antibodies were incubated at 4 °C overnight. Unbound antibodies were removed by washing in PBS containing 0.2% triton-X100 (PBST). Fluorophore conjugated secondary detection was used using the species-specific anti-IgG Alexafluor 488 antibodies from Life Technologies (A-21206 and A11055). These secondary antibodies were used at 1:400 and incubated with cells for 2h at ambient. Again, unbound antibodies were removed with gentle agitation with PBST washes. Nuclei were counter stained using Hoescht (1:500 dilution in PBST), and cells were mounted using AF1 mounting media. Confocal imaging was conducted using an Olympus FV100 microscope. Images were merged using ImageJ software.