Vessellike network organization in 3D culture assay. (A,B) Dissociated EBs (8 days old) were seeded on PLGA-PLLA scaffolds and incubated for 2 weeks. The scaffold was then fixed in formalin and immunostained with anti-PECAM1 (original magnification, × 100; 10×/0.3 NA oil-immersion lens). Micrographs were taken at 25°C using an inverted microscope (Axiovert 200, Carl Zeiss) equipped with an AxioCam colored camera. AxioVision 3.1 was used as imaging software. DAB was used as fluorochrome. (C,D) A vessellike network was formed during differentiation of the EBs. hEBs obtained on day 6 (panel C) and day 13 (panel D) were fixed and stained for anti-PECAM1 to exhibit the advancement in the vascularization process and formation of complex vascular network (original magnification, × 100; 10×/0.3 NA oil-immersion lens). Micrographs were taken at 25°C using a fluorescent motorized microscope (Axiovert 200, Carl Zeiss) equipped with an Orca digital camera (Hamamatsu). OpenLab and Fluoromount were used as imaging software and medium, respectively.