Image plus pro5

Following isolation, the aortas were cut longitudinally and fixed onto dishes. The aortas were stained with Oil-Red O for 30–40 min and then rinsed with 70% ethanol. Images of en face aortas were acquired using a camera. The sizes of the stained areas were calculated using Image-Plus Pro 5.0 (Media Cybernetics, Inc., Rockville, MD, USA).

The aortic arches were used to determine the area affected by atherosclerotic lesions. After the arches were dissected from the aorta, they were fixed in 4% paraformaldehyde overnight and embedded in paraffin. The embedded aortas were sliced into 5 µm-thick sections. The sections were then stained using hematoxylin and eosin (Wuhan Boster Biological Technology, Ltd., Wuhan, China). The average lesion size was determined using measurements obtained from 10 serial sections. Liver sections were stained with Oil Red O. All images were obtained with the inverted optical microscope (BX51; Olympus Corporation, Tokyo, Japan), The sizes of the stained areas were calculated using Image-Plus Pro5.0 (Media Cybernetics, Inc.).

The paraffin-embedded sections were deparaffinized and rehydrated. They were then processed for antigen retrieval in a microwave oven. The sections were then rinsed in PBS containing 0.1% Tween-20 (PBS-T) and blocked in 3% peroxide-methanol at room temperature to inhibit endogenous peroxidase activity. The sections were blocked in 3% bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.), for 1 h at room temperature and then incubated with primary antibodies for CD68 (dilution, 1:200) overnight at 4°C. Following this, the sections were then incubated with HRP-conjugated anti-rabbit secondary antibody (dilution, 1:100) for 50 min at room temperature, color was developed using 3,3-diaminobenzidine (Wuhan Boster Biological Technology, Ltd.). After the sections were counterstained with hematoxylin (Wuhan Boster Biological Technology, Ltd.), the slides were sequentially washed through an alcohol dehydration series and mounted using neutral gum. The slides were examined using the inverted optical microscope (BX51; Olympus Corporation), The sizes of the stained areas were calculated using Image-Plus Pro5.0 (Media Cybernetics, Inc.).