Tissue collection and morphological staining

To examine the atherosclerotic model and therapeutic effects of free SRT1720 and ICG/SRT@HSA-pept NMs, the total carotid artery and descending aorta were removed from mice after euthanasia, followed by several morphological staining protocols, including Oil Red O, H&E, Sirius Red, Masson, and immunohistochemistry staining. Several arteries (n=4) were frozen in Optimum Cutting Temperature (OCT) compound. Frozen aortic sections (8 μm) were stained with Oil Red O for ten minutes, followed by counterstaining with hematoxylin for two minutes at room temperature. Several arteries (n=6) were fixed in a 4% paraformaldehyde solution, embedded in paraffin, and sectioned at 5 mm for H&E and Masson staining. The sections were stained with hematoxylin-eosin (H&E) or Masson trichrome staining, and images of the stained sections were visualized using a light microscope (Olympus, Japan). The sizes of the plaque areas were quantified by delineating plaque areas in 3-4 root H&E stained sections/per mouse, and the data are presented as the mean plaque area. The necrotic core area was defined as the area that was negative for H&E staining within each plaque. The plaque composition was evaluated by the combination of necrotic core area and collagen fraction (Masson staining), according to previous reports ¹⁹.