Development of lymphatic endothelial cells in embryoid bodies.. Double-immunofluorescence stains of 27-day-old (A-F) and 21-day-old (G-O) EBs, cultured in the absence of VEGF-A or VEGF-C, for CD31-revealed (red; A,D) and Prox-1-revealed (green; B,E) CD31⁺ blood vessels and CD31⁺/Prox-1⁺ lymphatic vessels (C,F: merged images). Digital sectioning by laser confocal microscopy (D-F) revealed sprouting of lymphatic vessels (arrows) from preexisting blood vessels. Differential immunofluorescence stains for CD31 (red; G,J) and LYVE-1 (green; H) revealed that the CD31⁺/LYVE-1⁺ lymphatic vessels also expressed Prox-1 (green; K, arrows). (I,L) Merged images. Blood vessels expressed the vascular-specific marker MECA-32 (red; M), whereas Prox-1⁺ lymphatic vessels (green; N, arrow) were MECA-32^-. (O) Merged image. (J-L) Scale bars, 100 μm (A-L), 50 μm (M-O).