Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature. After blocking with 5% goat serum and 0.1% Tween20 in PBS for 1 hour at room temperature, cells were incubated with a primary antibody overnight at 4°C. After washing with PBS, cells were incubated with appropriate secondary antibodies for 1 hour at room temperature and observed under a confocal fluorescence scanning microscope (Nikon, Tokyo, Japan). The primary antihuman antibodies used were monoclonal antibodies for CD31 (DakoCytomation, Glostrup, Denmark), smooth muscle actin (Dako), Vimentin (Dako), and monoclonal antibody (Stro-1; Chemicon International, Herts, UK). Polyclonal rabbit antibodies used were against VEGFR2 (Santa Cruz Biotechnology, Santa Cruz, CA), von Willebrand factor (Sigma), pan-cadherin (Cell Signaling Technology, Beverly, MA), ZO-1 (Zymed Laboratories, San Francisco, CA), and β-catenin (Cell Signaling). Polyclonal goat anti-CD133 antibody (Santa Cruz Biotechnology) was also used. Respective anti-rabbit/anti-mouse Alexa Fluor 350, 488 and 568 IgGs (Molecular Probes, Invitrogen, Paisley, UK) were used as secondary antibodies.