Ab5731

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).

Alkaline phosphatase activity in biTBCs was measured using the Vector Alkaline Phosphatase Substrate kit (Vector, Burlingame, CA, USA). 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-Aldrich) and 0.1% Triton X-100 (Sigma-Aldrich) for 45 min at room temperature and then incubated overnight at 4°C with primary antibodies directed against OCT3/4 (1:50, SC-9081, Santa Cruz, Dallas, TX, USA), NANOG (1:250, AB5731, Millipore, Billerica, MA, USA), CDX2 (1:100, MU392A-UC, BioGenex, Fremont, CA, USA), or IFN-τ (1:1000, Operon, Tokyo, Japan). Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or 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-Aldrich).