Immunohistochemistry analysis of c-Kit(+) cells in bone marrow and muscle tissue surrounding femurs.

<p>Cryosections of femurs and surrounding muscle tissues from C57BL/6 mouse embryos at 14.5 to 19.5 dpc co-stained with c-Kit as an HPC marker and CD31 as an endothelial cell marker and observed by confocal microscopy. (A) Representative images showing hematoxylin-eosin (left) and immunohistochemical (right) staining of femur and surrounding muscle tissue at 16.5 dpc. Shown is c-Kit (green), CD31 (red) and TOTO-3 iodide (blue) staining. Scale bar represents 500 μm (left) and 50 μm (right). Green c-Kit(+) cells are observed in muscle tissue and indicated by arrows in right panel. (B) Confocal images of femur and surrounding muscle tissues at 14.5 dpc, 15.5 dpc and 16.5 dpc. Shown is c-Kit (green), CD31 (red) and TOTO-3 iodide (blue) staining. Scale bar represents 10 μm for all panels. (C) Localization of CD45(+) c-Kit(+) cells in serial sections (10 μm) of muscle tissue co-stained with antibodies against CD45 (red), c-Kit (green), CD31 (magenta) and DAPI (blue) at 14.5 dpc (left) and 16.5 dpc (right). Scale bar represents 10 μm for all panels. Arrows indicate CD45(+) c-Kit(+) cells, most located in muscle tissue rather than inside blood vessels. (D) The number of CD45(+) c-Kit(+) cells inside or outside of blood vessels in two sets of femur-surrounding muscle tissues was determined by microscopic observation of serial sections. Bars represent mean values and SD of cell numbers from three individual embryos. *<i>p</i><0.01.