Sudan IV Staining: A Procedure for Staining Lipid-Laden Atherosclerotic Plaques in Harvested Mouse Aortic Arch

Atherosclerosis occurs due to buildup of lipoproteins in the inner lining of the arteries, resulting in plaque accumulation, thickening and narrowing of the arteries, and reduced blood flow.

Plaques consist of a fibrous cap with smooth muscle cells and extracellular matrix, enclosing a core composed of low-density lipoproteins, LDLs - spherical particles with a hydrophobic core of cholesterol esters and triglycerides, surrounded by a phospholipid outer layer - along with inflammatory cells.

To visualize lipid-rich atherosclerotic plaques, obtain a freshly harvested mouse aortic arch. Transfer it onto a dark background-customized platform for improved contrast during wide-area imaging. Add buffer to prevent tissue dehydration.

Remove the perivascular adipose tissue surrounding the aortic arch to prevent false background staining. Incise the aorta and its branches longitudinally through the lumen to expose the innermost surface with atherosclerotic plaque buildup.

Secure the opened aortic arch onto the platform and incubate in ethanol. Ethanol displaces water from the tissue, inactivating proteins, and preserving the tissue architecture. 

Treat with Sudan IV stain solubilized in organic solvents. Within the plaque, Sudan IV, a fat-soluble diazo dye having high affinity for triglycerides and cholesterol esters of lipoproteins, moves from the organic solvent phase and binds to the LDLs’ hydrophobic core of lipids, forming stable complexes. Wash away excess stain using ethanol.

Under a stereomicroscope, the stained lipid-rich atherosclerotic plaques appear orange-red, while the non-plaque areas appear pale.