Confocal analysis of GFP-positive bone marrow (BM)–derived cells in the central nervous system of hematopoietic chimeras. Unless otherwise noted, images are brain sections; in all panels the green indicates the GFP signal. (A) Stereo pair image of a GFP-positive microglial cell folded around a capillary (red, isolectin). (B) BM-derived cell showing intracellular vacuoles positive for endothelial components (red, CD31; blue, isolectin). (C) Cross-section of a capillary with subendothelial phagocytic cells (red, CD31). (D-E) Elongated BM-derived perivascular cells (red, CD31; blue, 4′6-diamidino-2-phenylindole [DAPI]). (F) Retinal flatmount showing a BM-derived cell in contact with the astrocyte sheath surrounding a vessel (red, GFAP; blue, DAPI). (G) Bromodeoxyuridine (BrDU) incorporation in a brain capillary (red, isolectin; blue, DAPI). (G, inset) 3-dimensional digital rotation of the same image, showing that the BrDU-positive nucleus is adluminal (ie, endothelial). (H) BM-derived cells are located on the abluminal side of α-smooth muscle actin (α-SMA)–positive cells (red, α-SMA; blue, isolectin). (I) Detail from panel C. (J) Same staining as panel H at high magnification, showing that isolectin, α-SMA, and GFP are located on separate layers and do not colocalize. (K) Detail from panel F. (L) 3-dimensional digital reconstruction of a retinal capillary, showing that the GFP-positive cell is located on the outside of the astrocytic sheath surrounding the endothelial cells (red, isolectin; blue, GFAP). (M) CD11b (red) staining of BM-derived cells, also showing a CD11b⁺ GFP-negative cell (arrow). (N) CD45⁺ perivascular cell in a control mouse (red, CD45; blue, DAPI). (O) BM-derived perivascular cells are CD45⁺ (red, CD45; blue, DAPI). Bars: 10 μm.