Fig. 1. Expression of endothelial-specific markers during vascular development in EBs. (A) Immunoblotting was performed on total cell lysates of ES cells and EBs cultured for the indicated number of days, using antibodies against VEGFR-2, VE-cadherin and CD31. ß-catenin expression was analyzed as a loading control. (B) Immunohistochemical staining for VEGFR-2, VE-cadherin and CD31 in EBs cultured for the indicated number of days. One representative EB of eight for each condition is shown. Each condition was examined on two or more separate occasions. Arrows indicate blood islands visualized by VEGFR-2 and CD31 staining in day 4 EBs. Scale bars: 100 µm day 4 and 6; 200 µm day 8. (C) VE-cadherin was expressed in a 10-day old EB at the cell-cell contacts of the endothelial cells and the expression overlapped with CD31 expression. Scale bars: 100 µm.