PriCells: Immunohistochemistry of Postnatal human dental pulp stem cells (DPSCs) 

1. Primary DPSCs and BMSCs were subcultured into 8-chamber slides (2 × 10⁴ cells/well) (Nunc).
2. The cells were fixed in 4% formalin, and then reacted with saturating levels of primary antibodies and the corresponding control antibodies by using a broad-spectrum immunoperoxidase kit.
3. Antibodies used were: mouse (IgG) control; and rabbit (Ig) control, TUK4 (anti-CD14), QBEND 10 (anti-CD34), 2B11/PD7 (anti-CD45), M318 (anti-MyoD), 1A4 (anti-α smooth muscle actin), 2F11 (anti-neurofilament); H9H11 (anti-CD44), 6G10 (anti-VCAM-1); CC9 (anti-MUC-18); MAB1343 (anti-COL III), MAB1959 (anti-β1) (Chemicon); LF67 (anti-COL I), LF32 (anti-OC), BON-1 (anti-ON), LF100 (anti-BSP), LF123 (anti-OP); MAB1104 (anti-COL II); E-8 (anti-PPARγ), 147 (anti-FGF-2).
4. Working dilutions of rabbit serum (1/500), monoclonal supernatants (1/4), and purified antibodies (10 μg/ml) were used.
5. Secondary DPSC and BMSC cultures were washed in PBS and then fixed with 4% formalin.
6. Alkaline phosphatase activity was assessed by using a Sigma in vitro alkaline phosphatase substrate kit (85L-2). Calcium deposits were detected by treatment with 2% Alizarin Red S (pH 4.2).