Hematoxylin eosin

Frozen liver samples were sectioned at 10 μm and postfixed in 40% formaldehyde. Slides were then stained with Oil Red O (Sigma Aldrich) for 10 min, counterstained with Harris’s hematoxylin (PanReac AppliChem, Chicago, IL, USA) containing 4% acetic acid for 2 min, and finally cover-mounted for observation. Paraformaldehyde-fixed liver and WAT samples were routinely dehydrated, paraffin-embedded, and sectioned at 2–4 μm. Slides were then stained with hematoxylin-eosin (PanReac AppliChem, Barcelona, Spain) and cover-mounted for observation. Photomicrographs were obtained using an Olympus photomicroscope (Vanox AHBT3, Olympus, Tokyo, Japan) and Nikon (DS-Fi310, Nikon, Tokyo, Japan) digital camera. Adipocyte morphology (area and size) and hepatic steatosis degree (lipid-droplet total area, number per cell, and average area) were estimated using the NIH ImageJ v1.52p free software package (NIH, Bethesda, MD, USA). Data were collected from an average of 200 cells, two hematoxylin-eosin-stained sections per mouse. Histopathological changes were analysed blindly by two independent experimenters.

Morphological assessment of mouse brain tissue was carried out according to a published protocol [31 (link)]. In brief, the surgically isolated brains were fixed in 10% formalin solution and then incubated in sucrose solutions of increasing concentration (15% and 30%) for a total of 48–72 h. The samples were gradually filled with cryogel (Leica, Wetzlar, Germany) and cut into 10 µm coronal sections using a Leica CM1520 freezing sliding cryostat (Leica, Germany). The sections were stained with hematoxylin–eosin (PanReac AppliChem, Germany), and examined with a Zeiss Primo Star light microscope (Zeiss, Germany) with an integrated Axio CamMRc camera (Zeiss, Germany).

Mouse brains were surgically removed from the skull and placed in a 10% formalin solution at room temperature for 24 h. Next, the brain samples were incubated in a 15% sucrose solution (24–48 h) and then stored in a 30% sucrose solution for 24–48 h. Each brain was then placed in a Leica CM1520 freezing sliding cryostat (Leica, Wetzlar, Germany), progressively filled with Cryogel (Leica, Wetzlar, Germany) and sliced into 10 µm coronal sections. Every fifth brain section was mounted on a glass slide, air-dried for 24 h, and stained with hematoxylin–eosin (PanReac AppliChem, Darmstadt, Germany). The brain sections were dehydrated in increasing concentrations of alcohol, purified in xylene, and embedded in mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). The resulting samples were examined with a Zeiss Primo Star light microscope (Zeiss, Oberkochen, Germany) with an integrated Axio CamMRc camera (Zeiss, Oberkochen, Germany).