Figure 3. Bone sialoprotein staining of periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and F, cell cultures were stained with bone sialoprotein (green) and labeled for the Y chromosome (red dots). In panels B and D, bone sialoprotein staining was completed using DAB (brown). Bone sialoprotein staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express low levels of bone sialoprotein 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. (F) Mesenchymal stem cells (white arrows) express bone sialoprotein after co-culture (1:1) with periodontal ligament for 21 days. Panels A, C, E, and F are images taken from slides run in the same staining experiment and processed through the same solutions. (G) Negative control showing the DAB staining of periodontal ligament cells incubated with non-immune serum without bone sialoprotein antibody. (H) Histogram showing the mean optical density for mesenchymal stem cells (MSC) and periodontal ligament cells (PDL) and co-cultures after bone sialoprotein fluorescent staining. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.0001) as shown by ANOVA. Asterisk = P < 0.05, and double asterisk = P < 0.01 as compared with MSC. N 6. Bars = 50 µm (A, B, F) and 100 µm (C, D, E).