Immunocytochemistry evaluated the specific hepatic markers after the induction of differentiation. According to the protocol, the HLC-derived cells were rinsed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, 30525-89-4) for 15 min at 4°C, followed by 5 min at room temperature. After the fixative removal, cells were washed with PBS three times, and Triton X-100 was used to permeabilize cells in PBS. They were incubated with primary mouse monoclonal antibodies with a 1 : 200 dilution against α-fetoprotein (MAB1368; R&D) and albumin (MAB1455; R&D) overnight at 4°C. Next, the cells were rinsed with PBS-Tween 20 (0.1%) three times. They were also incubated with a secondary antibody, anti-goat mouse (1 : 100; F0102B; R&D), and phycoerythrin-conjugated [24 (link)] properly in the darkroom at 37°C for 60 min. Then, these matured cells were rinsed with PBS, and the nuclei eventually were counterstained with 4′, 6-diamidino-2-phenylindole ((DAPI) (1 μg/mL)) (Sigma-Aldrich, 28718-90-3) for 1 min. A fluorescence microscope (Nikon; Japan) was utilized to take images.
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