Oil red o solution

Oil red O staining was performed to determine the number of neutral lipids deposited in the liver, the method was described as Yin et al. (2021) [12 (link)]. Liver samples were firstly fixed with 4% paraformaldehyde and then frozen with liquid nitrogen. Secondly, the sectioned slides (6–10 μm thickness) were stained with oil red O solution (3 g L⁻¹, Servicebio, Wuhan, China) for 8–10 min. Then, the nuclei were counterstained with hematoxylin solution (Servicebio, Wuhan, China) for 3–5 min. After staining, the slides were washed and a cover glass was fixed to the slide with glycerol jelly mounting medium (Servicebio, Wuhan, China). The lipid droplets were observed using a light microscope (Nikon Eclipse Ni-U, Tokyo, Japan) at 100× magnification.

Liver tissues were weighed and homogenized. After centrifugation, the supernatants were measured for triglyceride using commercial kit (JianCheng Bioengineering Institute, Nanjing, China). Frozen sections of liver tissues were stained with Oil Red O solution (Servicebio, Wuhan, China) followed by hematoxylin staining.

Atherosclerotic lesion and lipid deposition in arterial lumina was assessed using Oil red O staining. Briefly, the intact aortas were opened longitudinally to expose the intimal surface, fixed in 4% paraformaldehyde overnight, stained with 0.5% oil red O solution (Servicebio, G1015, Wuhan, China) for 1 hour, and then differentiated in a 60% propylene glycol solution for 5 min. The stained aortas were photographed on a black background using a digital camera under identical light conditions and photographing parameters.

For HE staining, the liver tissues were divided and fixed in 4% paraformaldehyde for 24 h. After dehydration, the tissues were embedded in paraffin for sectioning. After the dewaxing process, the sections were stained with hematoxylin for 8–10 min. Sections were dehydrated again and were stained with eosin for 3 min. After dehydration, the sections were sealed with neutral gum. For Oil Red O staining, the liver tissues were fixed in 10% formalin and were prepared for frozen sections at a thickness of 6–10 μm. The sections were mounted on slides and were dried for 10 min at room temperature. The sections were incubated in 75% alcohol for 10 s and were stained in Oil Red O solution (#G1016, Servicebio). The slides were differentiated for 1 min until the background was colorless. After washing for 2 min, the sections were stained with hematoxylin for 8 min. The sections were washed and differentiated in a 1% aqueous HCl solution for 1 min. The sections were incubated in ammonia for several minutes. The slides were sealed with a glycerin gelatin solution.