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Fig. 8. Trans-endocytosis of CD47 from cultured hippocampal neurons to neighboring glial cells or to HEK293T cells expressing SHPS-1. (A-C) Mouse hippocampal neurons were cocultured for 1 hour with HEK–SHPS-1 cells that had been further transfected with an expression vector for GFP to allow visualization of cell shape. The cells were then fixed, stained in the presence of 0.1% Triton X-100 with a mAb to CD47 (red; A), and examined for GFP fluorescence (green; B). A merged image is shown in C. Arrows indicate CD47-positive vesicles in a HEK–SHPS-1 cell. Bar, 20 µm. (D-I) Mouse hippocampal neurons (and glial cells) were cocultured for 3 hour with HEK293T cells that had been transfected with an expression vector for CD47-YFP. The cells were then fixed and stained with mAbs to MAP2 (red; E) or to GFAP (red; H). Expression of CD47-YFP was confirmed by detection of YFP fluorescence (green; D, G). Merged images are shown in F and I. Arrows indicate CD47-positive vesicles in neighboring GFAP-positive cells. Bar, 20 µm. (J-L) Glial cells in mouse hippocampal neuron cultures were fixed and stained with mAbs to SHPS-1 (green; J) and to GFAP (red; K). A merged image is shown in L. Bar, 20 µm. (M-O) Hippocampal neurons (and glial cells) were isolated, cultured for 24 hours, and then transfected with an expression vector for CD47-YFP. Twelve hours after transfection, the cells were fixed and stained with a mAb to GFAP (red; N). Expression of CD47-YFP was confirmed by detection of YFP fluorescence (green; M). A merged image is shown in O. Enlarged images of the boxed regions are shown in the insets. Arrows indicate CD47-positive vesicles in a GFAP-positive astrocyte. Bar, 20 µm.
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