Figure 4. Osteopontin staining of periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and F, cell cultures were stained with osteopontin (green) and labeled for the Y chromosome (red dots). In panels B and D, osteopontin staining was completed with the use of DAB (brown). Osteopontin staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express high levels of osteopontin in vitro, as shown in panel C (green) and panel D (brown). In vitro co-cultures of mesenchymal stem cells and periodontal ligament (1:1) were incubated for 7 days (panel E) and 21 days (panel F). Periodontal ligament, mesenchymal stem cell, and co-culture images were from slides run in the same staining experiment and processed through the same solutions. (G) Negative control shows the DAB staining of periodontal ligament cells incubated with non-immune serum without osteopontin antibody. (H) Histogram showing the mean optical density for mesenchymal stem cells (MSC), periodontal ligament cells (PDL), and co-cultures after osteopontin fluorescent staining. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.0001), as shown by ANOVA. Double asterisk indicates that the OD for MSC staining was significantly less (P < 0.01) than that of the other 3 cell groups. N 6. Bars = 50 µm (F) and 100 µm (C, D, E). Panel C is the same magnification as A, and panel D is the same magnification as B.