Oil red o solution

The human umbilical vein endothelial cells (HUVEC, CRL-1730), human brain microvascular endothelial cells (HBMEC, CRL-3245), human aorta-vascular smooth muscle cells (HA-VSMC, CRL-1999), and human monocytes THP-1 (TIB-202) cell lines were purchased from the American Type Culture Collection (Manassas VA, USA); The oil red O solution was obtained from Beyotime Biotechnology (Shanghai, China); the shRNA lentivirus vector and the polybrene were obtained from the Cyagen Bioscience Inc., (Suzhou, China); IL-12, IL-6, and TNF-α ELISA kits were provided by Boster Biological Technology Co. Ltd. (Wuhan, China); Tissue Total cholesterol Assay Kit was from Applygen Technologies Inc. Beijing, China); PrimeScript RT reagent Kit, SYBR Premix DimerEraser™ (Perfect Real Time) assay kit, and the primers were purchased from Takara (Dalian, China); the primary antibodies p-TNK1 (D46E7), TNK1 (C44F9), p-STAT1 (58D6), STAT1 (D1K9Y), p-Tyk2 (Y1054), and Tyk2 (9312S) were purchased from CST (Danvers, MA, USA); the primary antibody for β-actin (AP0060) was purchased from Bioworld Technology (Nanjing, China); and the oxLDL was provided by the Peking Union-Biology, Co. Ltd (Beijing, China).

After being fixed in 4% paraformaldehyde at 23 °C for 10 min, the cells were rinsed thrice in 1% PBS. The plate was rinsed with washing solution for 30 s, oil red O solution (Beyotime, Shanghai, China) was added, and the plate was incubated at 23 °C for 30 min. The oil red O solution was washed away for 20 s using the washing solution. Cells were then immediately washed four times with 1 × PBS. Images were acquired under a microscope (Olympus, Tokyo, Japan) and analyzed using the Image J software (NIH, Bethesda, MD, USA).

The mouse aorta frozen section was fixed with 4% neutral buffered paraformaldehyde (Bmassay, Beijing, China) for 10 min and then rinsed with 60% isopropanol. The section was stained with Oil Red O solution (Beyotime, Shanghai, China) for 15 min. The nucleus was stained with Gill’s hematoxylin (Beyotime, Shanghai, China) for 30 sec. After rinsing with distilled water, the section was mounted in glycerine jelly (Bmassay, Beijing, China).