*2.12. Immunofluorescence of Cells in Culture*

HSCs were isolated and kept in culture as described above. Seven days after isolation, cells were incubated with serum-free medium containing either DMSO, 10 μM TGR5 agonist, or 10 μM forskolin. After 24 h, cells were washed with PBS and incubated with 5 μg/mL wheat germ agglutinin (WGA) (Thermo Fisher) labeled with AlexaFluor-594 in PBS for 10 min at 37 ◦C to stain glycoproteins or glycolipids within the outer leaflet of the plasma membrane [35]. Cells were fixed with ice-cold methanol for 3 min after removal of the WGA-solution. After washing of cells, unspecific binding was blocked using a solution of 5% FCS in PBS for 30 min. Activated HSCs were treated with either primary antibodies for Endothelin-A receptor (Abcam, Cambridge, UK) (1:500) or just blocking solution as negative control for 1 h. A secondary antibody labeled with AlexaFluor-488 (Dianova, Hamburg, Germany) (1:100) was used for detection of the primary antibody and incubated for 1 h. LSECs were fixed with methanol and incubated with antibodies directed against vascular endothelial Cadherine (Santa Cruz Biotechnology, Dallas, USA) (1:100) and TGR5 (Gpbar1 8/50, Roche) (1:20). Secondary antibodies labeled with Cyanine-3 (Dianova) (1:500) or fluorescein (Dianova) (1:100) were used at dilutions of 1:500 and 1:100, respectively. Intranuclear DNA was labeled by Hoechst 34580 (Thermo Fisher) (1:20,000). Pictures of LSECs were taken using the confocal microscope LSM 510 (Carl Zeiss, Jena, Germany). HSCs were imaged using a LSM 810 confocal laser microscope (Carl Zeiss, Jena, Germany). Colocalization analysis of murine hepatic stellate cells was carried out using Pearson's correlation coefficient calculated by the coloc2 plugin for ImageJ after selection of representative regions of interest ROIs in cell surface areas [33,34].
