In situ localization of CD271 and CD146 bone marrow cells. Immunofluorescence staining for the in situ localization of primary BM-MSCs. Paraffin sections of normal human bone marrow biopsies were stained with antibodies against CD271 and CD146. (A) Double-positive cells were detected as reticular cells surrounding the endothelium of vessels. Photographs in the left and middle panels illustrate staining against one antigen in double-stained specimens; these photographs are merged in the right panel. (B) CD271⁺ reticular cells, shown as green cells in the left panel and brown cells (black arrowhead) in the immunohistochemical (IHC) panels, were found as perivascular cells surrounding the endothelium, shown as red CD31⁺ cells in the left panel and black arrow in the middle IHC panel. (C) CD271⁺ bone-lining cells (white arrowheads in the left panel and black arrowhead in the IHC photograph) showed no expression of CD146 (green in the left panel). (B-C) Right IHC panels show a control for the CD271 staining using an isotype-matched antibody. IHC photographs were counterstained with hematoxylin. For confirming IHC expression analysis of CD146 on bone-lining CD271⁺ cells, see supplemental Figure 5. Nuclei were stained with TO-PRO3 (blue). Scale bars indicate 25 μm. Asterisk indicates the lumen of a vessel. Trabecular bone (b) is indicated. Immunofluorescence photographs were taken with a confocal microscope (DMRE; Leica); IHC photographs were taken with an upright microscope (BX51; Olympus).