Oro solution

For ORO staining, liver tissue samples were fixed using 4% paraformaldehyde in PBS of the liver, specimens were submerged in 15%, 20% and 30% sucrose solution per day. The samples were embedded in OCT compound (Tissue tek; Sakura Finetek USA) and sectioned into 20 μm slices. 3T3-L1 cells and liver tissues were washed with PBS and 70% isopropanol (Duksan Pure Chemicals), and stained with ORO solution (Sigma-Aldrich) at room temperature overnight. Samples were washed thoroughly with distilled water. Tissues were counterstained with Gill’s hematoxylin (Sigma-Aldrich). Images of the ORO staining were visualized with a bright field microscope (Nikon TE-2000U). To quantify the ORO staining intensities, image files were analyzed using ImageJ^{66 (link)}. First, original images of staining were converted into RGB images, and the red monochromatic image was used to calculate the area of the staining. “Threshold” tool in the “Adjust” box in the “Image” menu was used to adjust threshold manually. And then staining positive area was obtained by clicking “Measure” button under the “Analyze” menu.

Fat accumulation was determined by the weight of abdominal fat dissected from individual mice. Bodyweight measurements were taken before fat extractions. For Oil Red O (ORO) staining, frozen kidney sections were cut at 10 μm using a cryostat then air-dried for 30 min. ORO staining was performed by fixing the sections with 3 washes of 60% isopropanol, the incubating them with a working concentration (66%) of ORO solution (Sigma-Aldrich, St. Louis, MO, USA) staining for 15 min. Slides were then washed quickly with 60% isopropanol, quickly stained with Mayer’s hematoxylin to highlight nuclei, and then mounted using VectaMount AQ Aqueous Mounting Media (Vector Laboratories).

Cryosectioned liver samples and primary adipocytes were fixed with paraformaldehyde for 20 min and washed twice with distilled water. ORO solution (01391; Sigma-Aldrich) was added to the samples for 1 h at room temperature. The sections were washed with distilled water and counterstained with hematoxylin solution for 1 min whereas adipocytes were washed with PBS and incubated with 10 μM DAPI in PBS at room temperature for 10 min. Images were acquired and analyzed using a light microscope for tissue and fluorescence microscope for adipocytes. The slides were scored in a blinded manner.