Oil red o

HepG2 and AML12 cells were first incubated for 24 h with FFA, washed twice with
phosphate-buffered saline (PBS), fixed for 20 min with 4% formaldehyde, and thereafter
rewashed three times with PBS. The cells were then stained for 15 min using Oil Red O
(Nanjing Jiancheng Bioengineering Institute) and thereafter washed three times with PBS.
The resultant lipid droplets were observed and photographed under an electron microscope
(Zeiss).

4% Paraformaldehyde (PFA) was used to fix rabbit myocardial tissues, followed by paraffin embedding and tissue dehydration. Then, the tissue sample was cut into 5 mm thick sections. Cardiomyocyte architecture and interstitial fibrosis were assessed by hematoxylin and eosin (H and E) and Masson’s trichrome staining. The sum of the soft connective tissue areas was divided by the sum of the area of all types of connective tissues, and muscle area was calculated as collagen volume fraction (CVF). Accumulation of glycogen and lipid droplets in the cardiac tissue was detected by periodic acid-Schiff (PAS) staining, where glycogen was shown in red granules, and lipid droplets were visible in red color in Oil Red O staining (Nanjing Jiancheng, Inc, Nanjing, China). Images were visualized under an optical microscope at 400× magnification.

Liver tissues were sectioned and mounted on glass slides then stained with H&E. Each sample was observed at a 200× magnification of microscopic field in 10 randomly selected areas. The hepatic steatosis was evaluated by hepatic steatosis score, assigning from 0 to 3 where 0 = no steatosis, 1 = slight steatosis, 2 = moderate steatosis and 3 = severe steatosis. The liver cryostat section (8 mm) was stained with Oil Red O (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) staining.

The liver sections of mice were stained by Oil‐Red‐O (Jiancheng, Nanjing, China) to visualize LDs and the quantification was performed using IPP 6.0 software. The lipid content in cultured cells were also visualized and quantified using Oil‐Red‐O staining. In addition, lipids in the liver of mice were extracted and the hepatic TG and total cholesterol (TC) were measured using commercial kits (Jiancheng) according to the manufacturer's instructions.

Hearts were perfused with physiological saline via an injector inserted into the left ventricle (outflow via an incision in the right atria) and then frozen in OCT. Eight-micrometer cryosections were taken of the aortic root, and multiple plaques located in 5 sections in each mouse were stained with Oil red O (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The lesion area fraction was calculated by dividing the lesion area by the area of the aortic wall and expressed as a percentage.