Localization of ALCAM⁺ and Sca-1⁺ cells in BM. (A and B) Immunohistochemical staining of ALCAM (red), Sca-1 (green), and CD45, CD31 (blue) in adult mouse BM. CD45⁻/CD31⁻ ALCAM⁺ cells (arrowheads) and flat-shaped Sca-1⁺ cells (arrows) were localized to the endosteum. (B) Greater magnification of the enclosed box in panel A. Scale bar: 100 μm (A), 50 μm (B). (C-J) Immunohistochemical staining of ALCAM (red), Sca-1 (green), and OCN (blue) in adult mouse BM. Multiple views of the trabecular bone surface are shown. (C-F) Diaphysial region. (G-I) Metaphyseal region. A portion of OCN-positive osteoblastic cells expressed ALCAM on the bone surface in the trabecular area (arrowheads). In addition, flat-shaped Sca-1⁺ cells were also localized to the endosteal surface (arrows). Of note, there were no bone-lining nonhematopoietic cells that expressed both ALCAM and Sca-1. (D and F) Greater magnification of the enclosed box in panels C and E, respectively. (H and I) Greater magnification of the enclosed box in panel G. (J) Some of ALCAM⁺Sca-1⁺ hematopoietic cells (open arrowheads) adhered to the OCN⁺ALCAM⁺Sca-1⁻ cells (arrows) and OCN⁻ALCAM⁻Sca-1⁺ cells (arrow). Scale bar: 200 μm (G), 100 μm (C,E), and 50 μm (D,F,H-J).