IF microscopy

For standard IF staining, cells were cultured on coverslips coated with 5 μg ml⁻¹ fibronectin and subsequently fixed in 4% paraformaldehyde in PBS (supplemented with 1 mM CaCl₂ and 0.5 mM MgCl₂; PBS⁺⁺) for 10 min, permeabilized with 0.5% Triton X100 in PBS for 5 min and blocked for 30 min with 2% bovine serum albumin (BSA) in PBS. For the scratch assays HUVECs were cultured overnight. A scratch was made across the cell layer using a sterile pipette tip, and cells were allowed to recover and migrate for 4 h before fixation and IF staining. Primary and secondary antibody stainings were performed in 0.5% BSA in PBS for 1 h, and coverslips were mounted in Mowiol4-88/DABCO solution. For the IF stainings in Figs 7b,c and 8b,c,d,f, surface VE-cadherin was pre-labelled with an Alexa-Fluor-647-conjugated non-blocking anti-extracellular VE-cadherin antibody (mouse monoclonal 55/7H1) in EGM-2 medium at 37 °C for 30 min. After 1–2 h culturing these cells were fixed in paraformaldehyde and stained for surface VE-cadherin with mouse monoclonal 75 antibody (Fig. 7b,c) or indicated endogenous proteins. For the IF stainings in Fig. 9d, HUVECs were pre-labelled with 55/7H1 antibody in EGM-2 medium at 4 °C for 30 min and internalization was followed after 2 h of culturing the cells at 37 °C. To visualize intracellular proteins, VE-cadherin-labelled cells were permeabilized in 0.5% Triton X100 in PBS for 5 min and stained according to the standard IF protocol. Mesenteric arteries were stored in PBS⁺⁺ at 4 °C for no longer than 24 h after surgery until further preparation as described previously²⁵. IF stained samples were imaged using inverted Zeiss widefield microscopes Observer.Z1 equipped with a 63 × 1.40 Plan Apochromat oil objective, and Axiovert 200 equipped with a 63 × 1.25 numerical aperture (NA) EC Plan Neofluar oil objective, and AxioCAMMR3 and Hamamatsu Orca-R2 digital cameras. Confocal imaging of VE-cadherin and pacsin2 in HUVEC (Supplementary Movie 1), and human mesenteric arteries (Fig. 1f) were made by confocal laser scanning microscopy (Zeiss LSM 510) using a 63 × /1.4 NA oil objective and argon 488 and DPSS 561 nm lasers. For the imaging of VE-cadherin, pacsin2, Rac1 and F-actin in Fig. 3c, a LEICA TCS SP8 confocal microscope system equipped with a 63 × 1.4 NA oil objective, and 405 nm diode, 488 nm argon, 594 nm HeNe and 633 nm HeNe lasers, was used. Images were enhanced for display with an unsharp mask filter and adjusted for brightness/contrast in ImageJ (National Insitutes of Health).