Anti afp

The cells were fixed with 4% paraformaldehyde, and treated with PBS containing 5% normal goat or donkey serum, 1% BSA, and 0.2% TritonX-100. The following antibodies were used: SSEA3 (1:10), TRA-1-81 (1:50), TRA-1-60 (1:50) (these antibodies were used at Kyoto University and were kind gifts from Dr. Peter W. Andrews), anti-SSEA4, anti-TRA-1-81, anti-TRA-1-60 (1:500, all contained in the ES Cell Characterization Kit from Merck Millipore; these antibodies were used at Gifu University), anti-NANOG (1:20, R&D Systems), anti-OCT3/4 (1:1000, Santa Cruz Biotechnology), anti-βIII-tubulin (1:200, Cell Signal Technology), anti-βIII-tubulin (1:2000, Covance), anti-α-SMA (1:500, DAKO), and anti-AFP (1:100, R&D). Nuclei were stained using 1 μg/mL Hoechst33342 (Life Technologies).

Cultured cells were dissociated using dispase (Thermo Fisher Scientific) and a cell dissociation buffer (Thermo Fisher Scientific). Cells fixed with Cytofix Fixation Buffer (BD, USA) were washed with PhosFlow Perm/Wash Buffer I (BD) and incubated with primary antibodies (anti-HNF4α [GeneTex, USA], anti-CK19 [GeneTex], anti-AFP [R&D Systems, USA], anti-albumin [R&D Systems], anti-β-catenin [GeneTex], anti-Numb [R&D Systems], anti-Notch 1 ICD [R&D Systems], and anti-Hes5 [Bioss, USA] antibodies at a dilution of 1:400) at 4 °C overnight. After the cells were washed, they were incubated with Alexa 488 (Cell Signaling Technology, USA)- and phycoerythrin (Southern Biotech, USA)-conjugated anti-rabbit and anti-mouse IgG, respectively at 4 °C overnight. Then, the cells were washed again and passed through a 45-μm filter, and were subjected to flow cytometric analysis using a FACS Calibur (BD) flow cytometer according to the manufacturer's instructions. For flow cytometric analysis, the proportion of immunopositive cells was determined using the antibodies mentioned above. The data, which were normalized to the population of HNF4α⁺ cells, were represented as the relative value compared to that obtained following solvent control treatment.