Oil red o

The Oil Red O staining was performed as a previous study [13 (link)]. After fixing with 4% paraformaldehyde and washing with PBS, the aortas were treated with Oil Red O (Solarbio), which had been dissolved in isopropyl alcohol for 1 h. The samples were washed with 60% isopropanol and water and then photographed. The tissue separated from the aortic root was dehydrated with 15% and 30% sucrose and embedded in optimal cutting temperature compound. The samples were then frozen in liquid nitrogen and cut into 8 μM slices for Oil Red O (Solarbio) staining. The plaque area of the aortas and the lesion area of the aorta root were analyzed using Image J software.

Oxidized low-density lipoprotein (oxLDL) was used to develop an atherosclerotic cell model by stimulating lipid deposition in macrophages (or foam cell formation). In this experiment, the cells with no oxLDL stimulation and no nanoparticle treatment and the cells with oxLDL (100 μg/mL) stimulation but no nanoparticle treatment were recruited as a blank control group and a model group, respectively, whereas in the other groups the cells were stimulated/treated by both oxLDL (100 μg/mL) and nanoparticles. After incubating in a 5% CO₂ incubator at 37 °C for 24 h, the cells were washed three times with PBS, fixed with 2.5% glutaraldehyde for 20 min, washed three times again with PBS, and stained with Oil Red O by treating cells successively with 60% isopropanol for 3 min, Oil Red O (Solarbio, Beijing, China) for 20 min, and 60% isopropanol three times each for 1 min. After washing with PBS, the cells were observed by the inverted microscope, and the lipid deposition in cells (i.e., the ratio of the red area in cells to the total area of cells in an image) was analyzed by ImageJ software.

Transfected PTC cells were cultivated in a medium containing 100 µM oleic acid (OA) for 48 h. The cell lysates were incubated with reagents (37 °C for 30 min) from a TG Assay Kit (E1003, Applygen, China) before TG analysis. For Oil Red O staining, after incubation with oleic acid, the cells were fixed (4% paraformaldehyde), stained with Oil Red O (Solarbio, China) and counterstained with DAPI. Images were captured for subsequent analysis.

The liver specimens were embedded in O.C.T. (Tissue‐Tek) and cut into a thickness of 6 µm. For Oil Red O staining, the slides were incubated with Oil Red O (Solarbio) at 37℃ for 30 min, then used 60% 1,2‐propanediol to wash.¹⁹ For haematoxylin and eosin (H&E) staining, first, the slides were stained with haematoxylin for 5 min, then washed with 1% ethanol hydrochloride for 3 s. After water washing, the slides were incubated with eosin for 3 min and dehydrated with an alcohol gradient.²⁰

For BODIPY staining, cells were fixed in 4% paraformaldehyde for 15 min, permeabilized in PBS containing 1% Triton X-100 for 15 min, and stained with PBS containing BODIPY 493/503 (Invitrogen) and Hoechst 33342 (M&C) for 45 min at 37 °C. Analyses were performed with a High Content Analysis (Operetta).
For Oil Red O staining, cells were fixed with 70% ethanol for 10 min, stained with Oil Red O (Solarbio) in isopropanol for 30 min, and washed three times with 70% ethanol. For quantitative measurements of lipid accumulation, cells were washed with PBS to remove excess of stain solution, dried, and dissolved in 100% isopropanol. Absorbance was measured spectrophotometrically with FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 520 nm^{31 (link)}.

En-face Oil red O staining: aortas of two mice from each group were separated and fixed for 24 h. The vessels were cut longitudinally along the vessel wall and stained with Oil red O liquid for 60 min at 37°C. The vessels were then incubated with 75% ethanol until the atherosclerotic lesions turned red and the vessel wall became white. Cross section Oil red O staining: Aortic roots from six mice embedded in OCT were serially cross sectioned (5 μm thick sections) and stained with Oil red O (G1261-2, Solarbio, China) to evaluate the lesion size and lipid contents. After circling the outline of blood vessels (including whole valves), Image pro plus (Ver. 6.0, Media Cybernetics, MD, United States) was used to calculate the total area of blood vessels (including positive staining area) based on total area, and then the positive area is calculated based on Oil red O under the same contour, and finally the positive area/total area of blood vessels is calculated. Images were captured by Pannoramic DESK (3DHISTECH, Hungary) and preprocessed by CaseViewer (Ver. 2.4, 3DHISTECH, Hungary) and analyzed by Image-Pro Plus (Ver. 6.0, Media Cybernetics, MD, United States).