Immunofluorescent antibody staining with vascular markers in VEGF mutant ES cell cultures.

ES cells were differentiated, fixed on day 6 (G, G′), day 8 (A-F, I), or day 9 (H-H′), and stained with antibodies to ICAM-2 (A-C, G′, H′) or PECAM (D-G, H, I), followed by a PE-conjugated secondary antibody. (A, D, G, G′, H, H′),VEGF +/+ cultures; (B, E) VEGF +/− cultures; (C, F) VEGF −/−cultures; (I) −/−VEGF culture differentiated in the presence of 15 ng/mL recombinant hVEGF-165 from day 3 to day 8. The insets in D and F illustrate the differences in PECAM staining patterns, with arrowheads showing staining of borders between PECAM-expressing cells and arrows showing staining of borders between PECAM-expressing and nonexpressing cells. (G, G′) +/+ day 6 culture stained with PECAM (G) and ICAM-2 (G′) to show the lack of ICAM-2 staining before the development of patent vasculature. (H, H′) +/+ day 9 culture stained with PECAM (H) and ICAM-2 (H′) to show ICAM-2 staining of patent vasculature. Magnification, 50×.