Fast red substrate kit

Cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature and washed with PBS. Continuously, cells were treated with PBS containing 5% normal goat or rabbit serum (Nichirei Biosciences Inc., Tokyo, Japan) and 0.1% Triton X-100 at room temperature. Then cells were fixed and stained with antibodies to Oct4 (1/200 Millipore, Billerica, MA), Tra-1-60 (1/200, Stemgent^®, Cambridge, MA), Tra-1-81 (1/200, Stemgent^®), SSEA-1 (1/100, Stemgent^®), SSEA-4 (1/100, Stemgent^®), MAP2 (1/200, Millipore), Nestin (1/200, Millipore), α-smooth muscle actin (α-SMA: pre-diluted, DAKO Cytomation, Glostrup, Denmark), α-fetoprotein (1/100, R&D Systems Minneapolis, MN), and to the SeV nucleocapsid protein (mouse monoclonal antibody, clone #2E4, 1.6 mg/mL). These primary antibodies were visualized with Alexa Fluor^® 488-conjugated goat anti-rabbit IgG, or Alexa Fluor^® 488-conjugated rabbit anti-mouse IgG, or Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/200, Invitrogen, Carlsbad, CA). Cell nuclei were stained with 4', 6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Fluorescence images were taken using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss, GmbH, Germany). Alkaline phosphate (ALP) staining was performed using a Fast Red substrate kit (Nichirei) according to manufacturer’s instruction.

Alkaline phosphate (ALP) staining was performed using a Fast Red substrate kit (Nichirei Biosciences Inc., Tokyo, Japan) according to the manufacturer’s protocol as described previously (Yamasaki et al. 2014 (link)). Images of the dish were taken utilizing LUMIX (Panasonic, Osaka, Japan) and assessed for positive area using ImageJ (Abramoff et al. 2004 ). The reprogramming efficiency was determined as the number of ALP-positive colonies per total number of infected cells.