Prolong gold medium with dapi

Cells grown on coverslips were rinsed with PBS (pH 7.4) and fixed with 4% paraformaldehyde for 20 min at room temperature. Neutral lipids were stained by incubation with 1 μg/mL BODIPY 493/503 (Molecular Probes, Life technologies, Monza, Italy) in PBS for 30 min [38 (link)]. After washing, nuclei were stained and slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL) (ProLong Gold medium with DAPI; Invitrogen). Mounted slides were examined by Nikon Eclipse E80i light microscope (Nikon, Tokyo, Japan) equipped with the standard epifluorescence filter set up. Images were captured under oil with a 100× plan apochromatic objective. Analyses were performed on two independent experiments measuring at least 40 cells for each treatment using the ImageJ software [39 (link)].

Intracellular TG content was measured in FaO cells using the “Triglycerides liquid” kit (Sentinel, Milan, Italy), as previously described [19 (link),49 (link)]. Absorbance was recorded at 546 nm; after chloroform evaporation TG content was determined in which results were recorded spectrophotometrically. For measurement of extracellular TG content, the culture media were processed according to the same method. Values were normalized to protein content and data are expressed as percent TG content relative to controls [50 (link)]. For intracellular lipid staining, cells grown on coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Neutral lipids were stained by incubation with 1 μg/mL BODIPY 493/503 (Molecular Probes, Life technologies, Monza, Italy) in PBS for 30 min [40 (link)]. After washing, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL, (ProLong Gold medium with DAPI; Invitrogen). Mounted slides were examined at 10× magnification by Olympus IX53 light microscope (Olympus, Milano, Italy), equipped with the standard epifluorescence filter set up. Representative images were captured with a CCD UC30 camera and a digital image acquisition software (CellSens Entry).

Tissue slides were subjected to antigen retrieval with Tris buffer pH = 8.0 (Vector Labs H-3301) in a pressure cooker. They were then blocked in 5% normal donkey serum in phosphate buffered saline (PBS) containing 0.1% Triton-X, in combination with the avidin/biotin blocking reagent (Vector Labs SP-2001). Sections were incubated with primary and secondary antibodies and mounted in Prolong Gold with DAPI medium (Invitrogen). Biotinylated goat antibody (Jackson Immunoresearch 705-065-147) was applied to sections stained with TBX3, before detection with Streptavidin-647. GS and β-ACTIN staining was performed with the Mouse-on-Mouse detection kit (Vector Labs) according to the manufacturer’s protocol. The following antibodies were used: GFP (chicken, 1:500; Abcam ab13970), TBX3 (goat, 1:50; Santa Cruz sc-17871), GS (mouse, 1:500; Millipore MAB302), β-ACTIN (mouse, 1:100; Abcam ab8226), and HNF4α (rabbit 1:50; Santa Cruz sc8987). Samples were imaged at 20× magnification using a Zeiss Imager Z.2 and processed and analyzed with ImageJ software. For immunocytochemistry, plated cells were fixed with 4% paraformaldehyde, blocked in 5% normal donkey serum in PBS containing 0.1% Triton-X, and stained with primary and secondary antibodies as indicated above. Cells were imaged using a Zeiss Spinning Disk Confocal Microscope.