Immunofluorescence and analysis

Frozen liver sections were permeabilized using 0.3% Triton-X and incubated in antigen retrieval solution (Antigen retrieval citrate, Biogenex) at sub boiling temperature for 10 min. Subsequently, sections were incubated with blocking buffer containing 5% normal donkey serum (Jackson Immuno Research) followed by incubation overnight at 4 °C with mouse monoclonal primary antibody against HNF4 (1:800, Cat# PP-H1415-00, R&D Systems). Sections were washed and incubated with anti-mouse secondary antibody coupled with Alexa flour 488 (1:400, Invitrogen) or with DyLight 647 (1:400, Jackson Immuno) for 1 h at room temperature and counterstained with DAPI (40,6-diamidino-2- phenylindole, Sigma Aldrich). For lipid droplet staining, slides were incubated in Bodipy 500/510 (1:100 from 1 mg/ml, 4,4-Difluoro-5-Methyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid, Invitrogen) for 30 min. Slides were mounted using fluorescence mounting medium and images were obtained at 40x magnification using an Olympus IX71 fluorescence microscope. Fluorescence intensity of HNF4α-stained nuclei was calculated using MetaMorph TL software (version 7.6.5.0, Olympus).