Ve cadherin f8

Human VM‐derived ECs were seeded on gelatine‐coated coverslips in a way to reach confluency the next day and incubated overnight at 37°C in 5% CO₂. Then, cells were washed with warm PBS with Mg²⁺ and Ca²⁺ and then fixed with 4% PFA for 15 min at room temperature, followed by triple wash with PBS with Mg²⁺ and Ca²⁺. Cells were permeabilized with PBS containing 0.4% Triton X‐100 for 5 min and blocked with 2% BSA in PBS for 1 h at RT. The following primary antibodies were used for 1 h at RT: VE‐cadherin F8 (Santa Cruz, SC‐9989, 1:100), ERG (Abcam, ab92513, 1:400). Then, coverslips were washed three times with PBS for 5 min, followed by 45 min incubation at RT with appropriate secondary antibody in PBS: goat anti‐mouse Alexa Fluor‐488 (Invitrogen A11001, diluted 1:300) and goat anti‐rabbit Alexa Fluor‐568 (Invitrogen A11011, diluted 1:300). Then, coverslips were washed with PBS three times for 5 min, and in the last wash DAPI (Invitrogen, D1306, diluted 1:10 000) was added to visualize cell nuclei. Coverslips were mounted on a microscope slide in a mounting medium (ThermoFisher Scientific, 9990402).

Primary antibodies were purchased from the following sources and utilized at the following dilutions: VE-cadherin (F-8, Santa Cruz Biotechnology; 1:200), Arginase-1 (Sigma-Aldrich; 1:400), acetylated α-tubulin (6-11B-1, Santa Cruz Biotechnology; 1:100), and HNF4α (C-19, Santa Cruz Biotechnology; 1:400). Dylight 649-conjugated Ulex Europaeus Agglutinin I lectin (1:200) was purchased from Vector Laboratories. For secondary antibodies, Alexa Fluor 488, 568, 594, and 647 anti-mouse, anti-goat, and anti-rabbit IgG secondary antibodies were purchased from Life Technologies.