Immunofluorescence staining analysis of HFH.

HFH were harvested from 15 week gestational age HFL tissue and cultured on collagen type I-coated coverslips for 8 days in chemically-defined HCM. Cells were fixed with 4% paraformaldehyde and permeabilized and blocked with either PBS-T plus 1% gelatin (albumin detection) or PBS-T plus 1% BSA (for all other intracellular or cell surface markers). DAPI was also added to identify cell nuclei. The cultures were stained for albumin (A), CK8/18 (B), HepPar1 (C), HEA125 (D), CK19 (E), SMA (F), CD31 (G), or VE-cadherin (H). Unconjugated primary antibodies (all except CD31-PE and VE-cadherin-PE) were detected with a Cy3-labelled anti-mouse secondary antibody. HFH stained with secondary antibody alone served as a negative control (I). Photos were captured at a 63x magnification on a Zeiss fluorescence microscope using OpenLab Imaging Software (Improvision, Waltham, MA). Size bar indicates 15 µm.