Ms 113 p0

Alkaline phosphatase staining was performed using the Vector Alkaline Phosphatase Substrate kit (Vector). For immunofluorescence analysis, cells were fixed with PBS containing 3.7% paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were blocked with 5% bovine serum albumin (Sigma) and 0.1% Triton X-100 (Sigma) for 45 min at room temperature, and then incubated overnight at 4°C with primary antibodies against OCT3/4 (1:25, SC-5279, Santa Cruz Biotechnology), NANOG (1:250, AB5731, Millipore), glial fibrillary acidic protein (GFAP, 1:100, Z0334, DAKO), actin smooth muscle (ASM, 1:1000, MS-113-P0, Thermo), or alpha-fetoprotein (AFP, 1:100, MAB1368, R&D Systems). Alexa Fluor 594 goat anti-mouse IgG or IgM (1:500, Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (1:500, Invitrogen), Alexa Fluor 488 goat anti-mouse IgG or IgM (1:500, Invitrogen), and Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen) were used as secondary antibodies. Nuclei were stained with 1 μg ⁄mL Hoechst 33342 (Sigma).