Figure 3.

Col2.3GFPemd expression is restricted to alkaline phosphatase (AP)-positive cells near the bone surface or imbedded in matrix in teratoma bone formed by C341-6 cells. The presence of bone was shown by hematoxylin and eosin staining of the section after fluorescent imaging is completed (not shown). (A1): Bright green GFPemd-positive cells. No green fluorescent protein (GFP)-positive cells were detected in other parts of the teratoma. Scale bar = 200 μm. (A2): Alizarin complexone (AC) was injected 1 day prior to sample harvesting. Newly formed bone tissue is marked by AC labeling (red), and GFP-positive osteoblasts (green) were located adjacent to the AC labeling. Blue shows 4′,6-diamidino-2-phenylindole (DAPI) staining of cell nuclei. The boxed region is shown in B. (A3): The section shown in (A) and (B) was stained for AP activity, shown in yellow. DAPI staining again shows nuclei. (A4): Overlay of the images shown in (A2) and (A3). (B1): GFP and DAPI of the boxed portion of (A2). (B2): AC labeling was added to (B1). (B3): DAPI and AP (yellow)-positive cells were located adjacent to the AC labeling. (B4): Merged image of (B2) and (B3) shows that Col2.3GFP-positive cells are located in the band of AP-positive cells near the AC-labeled newly mineralized surface. AP is located primarily in the cell membrane and matrix vesicles that are released from the cells, whereas the GFP is cytoplasmic, so colocalization of the two signals is not precise. Because AP is expressed in preosteoblasts as well as mature osteoblasts, many of the AP-positive cells are not yet GFP-positive. The arrows indicate examples of GFP-positive cells that are also AP-positive. Scale bar = 20 μm. Abbreviation: B (in [A1] and [A3]), bone matrix.