Immunofluorescence Staining of Endothelial Cells

After 24, 48, or 72 h of incubation, samples were fixed in 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100 (Fisher Scientific, Waltham, MA, USA), blocked for 3 h with 4% bovine serum albumin (GE Healthcare Bio-Sciences, Chicago, IL, USA), incubated in primary (overnight) and secondary antibody (1 h), and mounted with FluoroGel II containing 4′,6-diamidino-2-phenylindole (DAPI) (Electron Microscopy Sciences, Hatfield, PA, USA) on glass slides. Primary antibodies used were anti-Glucose Transporter 1 (Glut-1) (ab15309, 1:200) (Abcam, Cambridge, MA, USA) and common endothelial markers such as anti-CD31 (89C2, 1:1000) (Cell Signaling Technology, Danvers, MA, USA) and VE-cadherin (2158, 1:100) (Cell Signaling Technology, Danvers, MA, USA) [35 (link),36 (link)]. With the immunostained samples, a Nikon Eclipse Ti inverted fluorescent microscope was used to acquire the wide-field epifluorescence images, while a Nikon C2 confocal microscope was used to acquire z-stacks of fluorescence images for 3-D reconstruction and analysis.