Anti hnf4

After the indicated treatment, the cells were fixed by the addition of 4% PAF, permeabilized with 0.1% Triton X-100 and incubated for 1 h at r.t. with the following primary antibodies: anti-HNF4 (1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), anti-CK8/18 (1:50 dilution; Cell Signaling Technology, Danvers, MA, USA), and anti-Vimentin (1:50 dilution; Cell Signaling Technology, Danvers, MA, USA). Unbound primary antibody was then removed, and the cells were washed with ice-cold PBS and incubated with DyLight 488 or DyLight 594 secondary antibody (1:500 dilution; Thermo Fisher Scientific, Waltham, MA, USA). Unbound secondary antibody was removed, cells were washed with ice-cold PBS, and nuclei were stained with Hoechst (Molecular Probes, Life Technologies, Carlsbad, CA, USA). In some cases, cells were stained with Actin Green (Thermo Fisher, Waltham, MA, USA) (1:125 dilution) to stain F-actin. Finally, the samples were analyzed by confocal fluorescence microscopy (Nikon A1 confocal microscope).