Figure 2. Osteocalcin staining and Y-chromosome tagging of human female periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and H, osteocalcin was fluorescently stained with SA-488 (green), and male mesenchymal stem cells were labeled for the Y chromosome (red dots). In panels B and D, osteocalcin staining was completed with DAB (brown). Osteocalcin staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express high levels of osteocalcin in vitro, as shown in panel C (green) and panel D (brown). (E) Co-cultures of mesenchymal stem cells and periodontal ligament (1:1) after 7 days in vitro. Images from panels A, C, and E were from slides run in the same staining experiment and processed through the same solutions. Negative controls included: (F) periodontal ligament cells not treated with osteocalcin antibody (i.e., non-immune serum) and stained; and (G) periodontal ligament cells incubated with antibody but not reacted with biotin-tyramide (reactant necessary for streptavidin SA-488 binding and the fluorescent green staining). (H) Cross-section confocal image of the interior (arrows) of cocultured cells after osteocalcin staining (green). Horizontal and vertical lines indicate the X and Y planes through which the cross-sectioned images were made. Note: Mesenchymal cell is tagged by a red dot. (I) Histogram showing the mean optical density values for various cell groups after fluorescent osteocalcin staining, including mesenchymal stem cells (MSC), periodontal ligament cells (PDL), and co-cultures of MSC/PDL after 7 or 21 days. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.05), as shown by ANOVA. Asterisk indicates that the OD for MSC staining was significantly less (P < 0.05) than for the other 3 cell groups. N = 3 for MSC and MSC/PDL (7 days), and N = 10 for PDL and MSC/PDL (21 days). Bar = 50 µm (A, C, and E). Panels B, D, F, G, and H have the same magnification as panel A.