Mab1455

Cells were fixed for 15 min at room temperature (RT) in 4% paraformaldehyde solution Roti-Histofix (Carl Roth GmbH & Co. KG), then washed three times in PBS (Life Technologies), and blocked and permeabilized for 1 hour in PBS with 1% fetal bovine serum (Sigma-Aldrich) and 0.1% of saponin (Sigma-Aldrich). Cells were then incubated with primary antibodies overnight at 4°C, rinsed with PBS, and incubated with secondary antibodies for 1 hour at RT. DAPI was used as a nuclear counterstain (Thermo Scientific). Antibodies used for characterization were alpha-fetoprotein (Dako A0008, rabbit polyclonal), HNF4a (Abcam ab92378, rabbit monoclonal), albumin (R&D Systems mab1455, mouse monoclonal), and cytokeratin-18 (Abcam ab82254, mouse monoclonal). To validate the efficiency, cells were cultured on two-well slides (Thermo Fisher Scientific Inc.) and after hepatic differentiation stained as described above for HNF4a and ALB. Whole slides (four wells in total) were scanned. The image analysis tool ImageJ (Schneider et al., 2012) was used to measure the area of double-positive cells.

For paraffin-section, tissues were harvested, fixed in 10% neutralized formalin for 2 days and then stored in 70% ethanol until further processing. For frozen-section, tissues were harvested, fixed in 4% paraformaldehyde (PFA) for overnight, submerged with 30% sucrose and then embedded into OCT compound. H&E staining and IHC were performed following standard protocols. The following antibodies were used for IHC: anti-GFP (Abcam, 6673, 1:400; Clontech, JL-8, 1:200); anti-Klf4 (Cell signaling, 3728, 1:200); anti-Ki67 (Cell signaling, 12,202, 1:200); anti-Sox9 (Abcam, 185230, 1:100); anti-Alb (R&D, MAB1455, 1:200); anti-BrdU (Abcam, 6326, 1:100); Phalloidin-488 (ThermoFisher Scientific, A12379, 1:400). (ThermoFisher Scientific, A12379, 1:400). After staining, the sections were mounted by DAPI Fluoromount-G (SouthernBiotech, 100–20) for nuclear counterstain.