Oil red o staining kit

Liver tissue samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 2 h, stored overnight in 10% formalin, and were embedded in paraffin. Cross-sections (5 µm) of the tissue were cut and used for staining with hematoxylin and eosin. To evaluate the degree of hepatic lipid accumulation, oil red O staining of the sections was performed using an oil red O staining kit (Nanjing Jiancheng Bioengineering Institute) following the manufacturer's instructions. Images were taken using a light microscope. The NAFLD activity score (NAS) were performed by an experienced pathologist without prior knowledge of the treatments. The scoring includes measurement of steatosis grade (0-no steatosis, 1- < 50% steatosis, 2-> 50% steatosis, 3- > 50% steatosis + microvesicular steatosis), hepatocyte ballooning (0-none, 1- < 66%, 2- > 66% hepatocyte involvement) and inflammation (0-no foci, 1- < 2 foci, 2–2–4 foci, 3-+4 foci). NASH was defined in the cases of NAS of ≥5 (Kleiner et al., 2005 (link); Zhang et al., 2016 (link)).

Liver tissue was fixed in liquid nitrogen, embedded in ornithine carbamoyl transferase (OCT) medium and sectioned at − 20 °C at a thickness of 10 μm. The sections were stained using an Oil red O staining kit (lot number 20180528, Nanjing Jiancheng Bioengineering).

Cell counting kit-8 (CCK-8) was from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Oil red O staining kit, FFA detection kit and TG assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Glycerol content GPO-POD enzymatic assay kit was purchased from Applygen Technologies Inc, China. ELISA kits for quantitative analysis of mouse TNF-α, IL-6, adiponectin, leptin and FAS, rabbit anti-IR-α, goat anti-rabbit IgG (γ-chain specific) and goat anti-mouse IgG (γ-chain specific) were from Wuhan Boster Biotech Co., Ltd (Wuhan, China). Insulin, 3-isobutyl-1-methylxanthine (IBMX), DXM and antibody for mouse GLUT4 were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). Antibodies for IRS1, phospho-IRS1 (Ser307), PI3K/p85, phospho-PI3K/p85 (Tyr458)/p55 (Tyr199), Akt (pan), phospho-Akt2 (Ser473) and phospho-AS160 (Thr642) were purchased from CST China (Shanghai, China). Enhanced BCA Protein Assay Kit, Cell lysis buffer, PVDP membrane (0.45 μm), BeyoECL Plus were purchased from Beyontime Institute of Biotechnology (Shanghai, China). Rosiglitazone was provided by Guangzhou Institute for Drug Control (Guangzhou, China).
6α-Hydroxylup-20(29)-en-3-on-28-oic acid (1) was obtained from Viburnum odoratissimum in our laboratory [11 (link)]. It was dissolved in DMSO (20 mmol/L) and diluted with cell growth medium for experimental usage.

HE staining was performed on 4-μm-thick sections according to the standard published protocols [23 (link)]. Sections were examined by experienced liver pathologists. The NAFLD activity score (NAS) was determined as described previously according to the following score [24 (link)]: steatosis (0 = < 5%; 1 = 5–33%; 2 = 33–66%; 3 = > 66%); intralobular inflammation (0 = no lesions; 1 = < 2 lesions/field of view; 2 = 2–4 lesions/field of view; 3 = > 4 lesions/field of view; and ballooning degeneration (0 = none; 1 = rare new balloon cells; 2 = common new balloon cells).
Frozen liver tissues were sectioned at 10-μm thickness on a cryostat (Leica CM1850, Germany), and fat cells were detected by an oil red O staining kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China; batch number: 20180704) following the manufacturer’s instructions. Next, sections were analyzed under an inverted fluorescence microscope (Leica 37XB, Germany) and photographed.

The cells were washed with PBS twice and fixed with 10% paraformaldehyde for 30 min. Oil red O staining was performed using an Oil red O staining kit (#D027, Jiancheng Biotech, China) according to the manufacturer’s protocols.