Confocal fluorescence microscope

Brains were frozen to investigate the localization of Nrf2 and Keap1 using immunofluorescence (IF) staining. The brain sections were incubated with rat anti‐Nrf2 (1:200, D9J1B, CST) and rabbit anti‐Keap1 antibodies (1:200, D6B12, CST) at 4 °C overnight. The sections were then incubated with goat anti-rabbit (1:500, CSA3611, Cohesion) and goat anti-rat (1:500, CSA3229, Cohesion) secondary antibodies at room temperature (22–25 °C) for 1 h. Finally, the sections were incubated with DAPI (1 ng/μL, Solarbio) for 5 min at room temperature (22–25 °C). Fluorescent signals were detected using a confocal fluorescence microscope (Olympus).

For the tissue immunofluorescence staining, a 10-µm thick section was fixed in acetone at 4 ℃ for 15 min, and then blocked with 5% BSA containing 0.4 % Triton X-100 and incubated for 30 min at room temperature. After incubation with primary antibodies at 4 ℃ overnight, the tissue slides were incubated with fluorescence (Alexa Fluor 488 or 594)-conjugated secondary antibodies (Invitrogen, USA) in the dark for 1 h at room temperature. Next, the tissue slides were incubated with 1 µg/mL 4’,6-diamidino-2-phenylindole (DAPI; Thermo Scientific, USA) for nuclear labeling. For the cell immunofluorescence staining, the cells were fixed by 4% paraformaldehyde for 10 min. After blocking with PBS containing 10% FBS, the cells were then incubated with primary antibodies at 4 ℃ overnight. After being rinsed 3 times with PBS with 0.2% Triton X-100, the cells were incubated with fluorescence-conjugated secondary antibodies (Invitrogen, USA) in the dark for 1 h. All the samples were observed under a confocal fluorescence microscope (Olympus, Shinjuku, Japan).

For aortic sinus analysis, hearts were fixed in 4% formalin, dehydrated, embedded with paraffin, and sliced into 5-μm-thick sections from the aortic region towards the apex of the heart. H&E staining (Servicebio) was then performed. For plaque analysis, fibrous caps and necrotic cores were examined using Masson’s trichrome staining (Servicebio). To measure fibrous cap thickness, at least three measurements of the thinnest fibrous cap within one atherosclerotic plaque were taken and averaged, as described previously.^{14 (link)} The necrotic core area was analyzed by measuring the total acellular area in atherosclerotic plaque, as described previously.^{14 (link)} F4/80 staining was performed to determine the macrophage content in the atherosclerotic plaque. Sections were stained with anti-F4/80 antibody (1:500, rat anti-mouse monoclonal antibody) followed by incubation with goat anti-rabbit (1:300) antibody. Slides were mounted with mounting medium containing DAPI (Servicebio). Images were analyzed in the area of atherosclerotic lesions using a confocal fluorescence microscope (Olympus, Tokyo, Japan) and Image-Pro Plus software (Media Cybernatics).