Ab82254

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.