Histological Analysis of Atherosclerotic Plaques

For the histological analysis of atherosclerosis plaques, dissected hearts were fixed in 4% PFA for 4–5 hours and incubated in 30% sucrose at 4 °C overnight. Fixed hearts were horizontally cut, and the half containing the apex was frozen in optimal cutting temperature (O.T.C.) embedding medium (Sakura Finetek, Japan). Serial sectioning was performed in the area of the aortic root by cryotome (Leica, Germany). Each section was 6 µm thick. Hematoxylin and eosin staining and ORO staining were performed as described in established protocols^{3 (link),14 (link)}. For immunohistochemical analysis of Mac2 expression in the plaque macrophages, frozen sections were incubated with a 1:200 dilution of rat anti-mouse Mac2 antibody (Cedarlane, Canada). For secondary fluorescent staining, a 1:500 dilution of Alexa Fluor 488-conjugated chicken anti-rat IgG secondary antibody was used (Invitrogen, USA). Stained histological sections were observed with a BioRevo fluorescence microscope (Keyence, Japan). iRFP fluorescence expression was observed in consecutive, unstained sections under a Cy5.5 filter using a fluorescence microscope (Olympus, Japan).