Fig 1: Successive steps for obtaining fluorescent immunostained microscopic images of the GLUT1 signal in a cross section of a capillary in a mouse cerebral cortex. (a) Snapshots captured with a 40× objective lens were tiled to cover the entire hemisphere. Scale bar = 1 mm. (b) A region of interest (ROI; white square) was set in the integrated light image (14 901 × 16 778 pixels, 0.4 µm/pixel). Scale bar = 1 mm. (c) CLSM images of the ROI with 21 XY planes at 1-µm intervals were simultaneously captured. (d) One CLSM image including a cross section of a BCEC was chosen, and this BCEC cross-section was set as a new square ROI (0.262 µm/pixel, 4622 × 4622 pixels). Scale bar = 200 µm. (e) After laser marking, CLSM images of the new ROI with 44 XY planes at 1-µm intervals were obtained. (f) A single optical section including the target BCEC cross section and laser marking is shown. Scale bar = 50 µm. (g) A representative CLSM image of a BCEC cross section is shown. Scale bar = 10 µm. The green fluorescence represents anti-GLUT1 antibody/Alexa Fluor 488 FluoroNanogold-Fab’ anti-Rabbit IgG, whereas the blue represents 4',6-diamidino-2-phenylindole (DAPI; a marker of nuclear DNA).
Fig 2: Pre-absorption test of anti-GLUT1 antibody by immunohistochemistry. (a) Capillary vessel walls in a mouse brain were stained with the antibody. (b) Intensity of the GLUT1 immunostaining in the capillary was greatly decreased after pre-incubation with GLUT1 peptide. The inset images are a higher magnification of the rectangles in the main image. Scale bars = 50 µm in main image; 5 µm in inset image.
Fig 3: Effect of anti-GLUT1 antibody concentration on the amount of immunoEM signals. Without the primary antibody, GLUT1 signals were rarely observed (a, d). Electron micrograph showed that, at higher primary antibody concentration, labeling of GLUT1 appeared to be enhanced on the luminal side of BCECs (b, e). On the other hand, at a lower primary antibody concentration, labeling appeared to be confined to the cytoplasm and plasma membrane of BCECs. Scale bars = 1 µm in (a–c), 500 nm in (d–f).
Fig 4: Pre-absorption test of polyclonal anti-GLUT1 antibody by western blot analysis. The GLUT1 band was strongly detected in normal mouse brain lysate with anti-GLUT1 antibody (left lane). The band was not detected after pre-incubation of the anti-GLUT1 antibody with GLUT1 peptide (right lane).
Fig 5: Detection of GLUT1 molecules by light microscopy and transmission electron microscopy. (a) An optical section (2.18 µm thick) was acquired by CLSM, as shown in Fig. 3g. The section contained nucleus (blue) and GLUT1 (green) signals. Scale bar = 10 µm. (b) GLUT1 fluorescence signal spreading in the Z direction in a capillary vessel is illustrated. (c) Serial sections (80 nm thick) of the optical section were obtained. (d–i) Electron microscopic images of a capillary cross section showed GLUT1 molecules in the blood capillary endothelial cells (arrowheads). Each panel shows a different serial section; the sections displayed are not consecutive. Scale bars = 1 µm. (e, g, i) Higher magnification of the squares in (d, f, h). Scale bars = 1 µm.
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