Immunocytochemical Staining of MVECs

MVECs were grown on collagen IV/fibronectin-coated tissue-culture ware or 12 mm Costar Transwell filters. Cells were fixed using 3.7% formaldehyde and extracted in acetone (−20°C). Alternatively, they were fixed and permeabilized simultaneously in 80% MeOH, 3.2% formaldehyde, 50 mM HEPES (pH 7.4) (Martins et al., 2013 (link)). Staining was performed as previously described (Turowski et al., 2004 (link)) using antibodies against von Willebrand factor (Dako), VE-cadherin (Martins et al., 2013 (link)), occludin and Cldn5 (Invitrogen), VEGFR1 (sc-31173; Santa Cruz Biotechnology), VEGFR2 (ab11939; Abcam), and P-glycoprotein (clone C219).