Cm1800

Formalin-fixed and alcoholic dehydrated liver sections were embedded in paraffin, cut into 5 μm sections, and stained with H&E. The sectioned tissues were examined for morphological analysis and histological scoring of steatosis, hepatocellular ballooning, and lobular inflammation by light microscopy [31 (link)]. To assess lipid accumulation, frozen liver specimens were cut into 10 μm sections using a cryostat (LEICA CM1800) and then fixed and stained with ORO stain [32 (link)]. Images were taken randomly from each tissue section using XSZ-07 series of a biological microscope (China) and Apex Minigrab (UK). The ORO-stained sections were automatically analyzed using Image J (https://imagej.nih.gov/ij).

A portion of the liver and adipose tissue was fixed with a 10-fold volume of 10% formalin. Thereafter, the tissues were embedded in paraffin blocks and sectioned using a cryo-cut microtome (Leica CM1800, Wetzler, Germany) with a thickness of 3–4 μm. The sections were stained with hematoxylin and eosin (H&E) and captured using an optical microscope (ZEISS Axio Imager 2, Carl Zeiss, Oberkochen, Germany). The liver histological changes were assessed (by a blinded observer) in three different randomly selected 20X fields for each experimental rat. Histological scores were measured using Kleiner’s histological scoring system [55 (link)] by quantifying the degree of inflammatory cell infiltration, steatosis, and balloon cells. Adipocyte size (μm²) and the number of crown-like structures (CLS) were determined in three different randomly selected 20X fields for each experimental rat. Adipocyte size and CLS number were measured using an Image J (NIH, Bethesda, MD, USA).

The mice were anesthetized with pentobarbital sodium (100 mg/kg, i.p.) and transcardially perfused with 50 mL of 0.01 M phosphate buffer saline (PBS, pH 7.4) and thereafter fixed with 4% paraformaldehyde (pH 7.4). The brains were then collected and subsequently immersed in 0.1 M PB containing 30% sucrose at 4 °C. Coronal sections via the SNr area with a thickness of 30 μm were sectioned on a cryostat (Leica CM1800; Heidelberg, Germany). Sections were incubated in a blocking PBS buffer containing 0.3% Triton X-100 and 0.05% sodium azide, 10% bovine serum albumin for 30 min at room temperature. The primary antibodies included mouse anti- Mouse anti-GAD67 (GAD1) (1:100, Cat. #ab26116, Abcam), Rabbit anti-GAD65 (GAD2) (1:50, Cat. #5843 s, CST), Rabbit anti-Parvalbumin (1:200, Cat. #80561 s, CST), Rabbit anti-vGlut1 (1:500, Cat. #a12879, Abclonal), Rabbit anti-GFAP (1:200, Cat. #12389 s, CST), and Rabbit anti-NeuN (1:50, Cat. #ab190565, Abcam). The secondary antibodies Alexa 488-Goat anti-Mouse (1:500, Cat. #A23210, Abbkine) and Alexa 488-Goat anti-Rabbit (1:500, Cat. #A23220, Abbkine) were used, together adding DAPI (1:500, Cat. #BMD0063, Abbkine) at room temperature for 12 min. All images were taken under FV-1000 confocal fluorescence microscope. There were 3 mice in each group and at least 4 slices were taken from each brain tissue.