Suitable secondary antibodies

Plated cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. Cells were washed three times with PBS after each step and then blocked with 5% bovine serum albumin (Nacalai Tesque) for at least 1 h at room temperature. The cells were permeabilized prior to primary antibody incubation. Cells were then incubated with primary antibodies at 4 °C overnight and washed three times with PBS. The following primary antibodies were used in this assay: NANOG (1:500; ReproCELL, Yokohama, Japan), SSEA4 (1:1,000; Chemicon, Darmstadt, Germany), βIII-tubulin (1:2,000; Covance, Princeton, NJ), MAP2 (1:1,000; Millipore, Billerica, MA), SMI-32 (1:2,000; Covance), GFAP (1:1,000; DAKO, Glostrup, Denmark), TFG (1:1,000; Protein Tech, Rosemont, IL), Ubiquitin (1:1,000; DAKO), FK2 (1:1,000; MBL, Nagoya, Japan), SOX17 (1:1,000; R&D Systems, Minneapolis, MN), and αSMA (1:500; DAKO). Suitable secondary antibodies (Invitrogen) were incubated with samples at room temperature for 1 h, and washed three times with PBS. Nuclear staining was also performed with 0.5 g/mL 4′,6-Diamidino-2-Phenylindole (DAPI) for 5 min, and then the cells were washed three times with PBS. Coverslips were mounted in ProLong Gold antifade reagent (Invitrogen). Images were collected using LSM710 (Carl Zeiss, Oberkochen, Germany) or IN Cell Analyzer 6000 (GE Healthcare).