Characterization of 3D hAR cultures. (A) hARs sprout in the presence of EGM-2 but only after fibroadipose tissue cleaning. Representative micrographs of hARs with or without fibroadipose tissue cleaning, cultured with 5% FCS EBM-2 (Basal medium) or with EGM-2 (complete medium). The bottom micrograph is a magnification of the red dotted line square. Scale bar represents 200 μm. (B) 3D isosurface rendering of hAR sprouting of whole-mount immunofluorescence staining for VE-cadherin (red) and nuclei (blue) acquired through confocal microscopy. Tickmarks on axis represent 50 μm. (C) Confocal images of hAR sprouts treated with Ac-LDL or Dextran in green and stained with anti-VEGFR2 Ab in red and with DAPI in blue. Scale bar represents 10 μm. (D) Sprout lumen is shown by 3D rendering of magnification of (B) and z-stack section of yellow dotted line rectangle of hAR sprouting outgrowth stained for VE-cadherin (red) and nuclei (blue). The yellow arrow indicates the capillary lumen. Scale bar represents 5 μm. (E) Pericytes coverage. Immunostaining of whole-mount hARs with anti-VEcadherin (red), NG-2 (green), and nuclear staining with DAPI (blue). Scale bar represents 10 μm.