Antigen retrieval citrate

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 anti-HNF4α monoclonal antibody (1:800, Cat# PP-H1415–00, R&D Systems), rabbit polyclonal anti-VDAC antibody (1:400, cat# PA1–954A, Invitrogen) and cleaved caspase3 antibody (1:500, cat#9664, Cell Signaling). Sections were washed and incubated for 1 h at room temperature with anti-mouse secondary antibody coupled with Alexa fluor 488 (1:400, Invitrogen) and anti-rabbit secondary antibody coupled with rhodamine red. Nuclei were visualized by counterstaining with DAPI (40,6-diamidino-2-phenylindole, Sigma Aldrich). Slides were mounted using fluorescence mounting medium and images were obtained at ×40 magnification using an Olympus IX71 fluorescence microscope. Fluorescence intensity of HNF4α-stained nuclei and VDAC stained mitochondria was calculated using MetaMorph TL software (version 7.6.5.0, Olympus). Fold change was calculated by normalizing to values from mice fed NC.

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).

Frozen intestine sections were permeabilized using 0.3% Triton-X and incubated in antigen retrieval solution (Antigen retrieval citrate, Biogenex) at sub boiling temperature for 10min. Subsequently, sections were incubated with blocking buffer containing 5% normal donkey serum (Jackson Immuno Research) followed by incubation overnight at 4°C with primary antibody against HNF4α (1:800, Cat# PP-H1415-00, R&D Systems) or Lysozyme (1:200, Cat# PA1-29680, Invitrogen). Sections were washed and incubated with anti-mouse or rabbit secondary antibody coupled with Alexa flour 488 (1:400, Invitrogen) or with Rhodamine Red (1:400, Jackson Immuno) for 1 hour at room temperature and counterstained with DAPI (40,6-diamidino-2- phenylindole, Sigma Aldrich). 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).