Las core v4

Samples of liver and adipose tissues (subcutaneous, epididymal, retroperitoneal and brown) were immediately fixed in 10% formaldehyde, embedded in paraffin and cut into 4 μm (liver and BAT) or 6 μm (WAT) slices. For each sample, two sections were stained with hematoxylin and eosin (H&E). The samples were observed under a microscope (Leica DM750 Wetzlar, Germany), photographed with a digital camera (Leica DMC2900), and processed with the imaging software Leica LAS Core V4.5. Analysis of adipocyte area of at least 100 adipocytes per section was done using the Adiposoft software for Image J (ImageJ, NIH)41 (link). To visualize the hepatic neutral lipids, frozen liver tissues were sectioned with a cryostat (8 μm) and stained with Oil Red O (ORO) at 0.5% in propylene glycol (Sigma-Aldrich, St. Louis, MO, USA). For the quantitative analysis of the ORO staining, images were converted to an 8-bit grayscale in ImageJ as described42 (link) and the integrated density was measured, which is the product of area and mean gray value.

2 mice/group were randomly sacrificed on day 3 of treatment, and all others at day 21. Tissue samples were immediately fixed in a 10% formaldehyde solution. Each tissue was embedded in paraffin, cut into 5 μm slices, and stained with hematoxylin and eosin (H&E). The samples were blindly analyzed by a pathologist with a microscope (Leica DM750, Wetzlar, Germany), photographed with a digital camera (Leica DMC2900), and processed with Leica LAS Core V4.5.