Immunocytochemistry d7EB cells were fractionated using FACS according to their cell surface expression of ACE and KDR and were subcultured on 35-mm fibronectin-coated dishes on top of No. 1.5 coverslips (0.16Ϫ0.19 mm). After 2 weeks, the cells were washed twice with PBS, fixed with paraformaldehyde 4% in PBS for 30 minutes, washed with 100 mmol/L of glycine in PBS, and incubated for 1 hour with blocking buffer containing 2% to 5% normal donkey serum, 0.2% bovine serum albumin, and 0.1% Triton X-100. Subsequently, they were incubated for 2 hours at room temperature with primary antibodies against human vascular endothelial cadherin, von Willebrand factor, and CD43 at 1:200 dilution in 2% normal donkey serum, 1% bovine serum albumin, and 0.1% Triton-X in PBS. Control cells were incubated with goat IgG, rabbit IgG, and mouse IgG 1 , respectively, under the same conditions. After washing with PBS and repeat blocking for 1 hour, the cells were incubated with secondary antibodies Alexa 488 anti-goat IgG, Alexa 568 anti-rabbit IgG, or Alexa 568 anti-mouse IgG1 at 1:700 dilution for 1 hour at room temperature.