Carotid (LSS and OSS regions) and aortic sinus plaques were serially cut in 7 μm transversal sections and stained as previously described [31 (link)]. Eleven sections per staining (separated by 45 μm from each other) from each mouse specimen were fixed in acetone and immunostained with the following specific antibodies: anti-ACE2 (dilution 5 μg/mL, Abcam, Cambridge, UK), anti-mouse CD68 (macrophages, dilution: 1:400; ABD Serotec, Dusseldorf, Germany), anti-mouse Ly6G (neutrophils, dilution: 1:100; BD Pharmingen™, San Jose, CA), anti-mouse MMP-9 (dilution: 1:60; R&D Systems), anti-mouse Ly6B.2 (neutrophils, dilution: 1:200; AbD Serotec, Kidlington, UK), rabbit anti-mouse ADAM-17 (dilution: 10 μg/mL; Abcam, Cambridge, UK), rat anti-mouse ICAM-1 (dilution: 1:200; Abcam, Cambridge, UK) and rabbit anti-mouse VCAM-1 (dilution: 1:200; Abcam, Cambridge, UK)
For the images obtained in brightfield microscopy, the quantifications were performed with the MetaMorph software and the data were calculated as percentages of stained area from the total lesion area (macrophages and MMP-9) or number of cells per millimeter squared (neutrophils). Fluorescent images were obtained on a confocal microscope equipped with a digital imaging system (Carl Zeiss LSM 700, Zeiss, Baden-Württemberg, Germany) and fluorescence intensity was quantified by Image J software (NIH, Bethesda, MD, USA).
For the images obtained in brightfield microscopy, the quantifications were performed with the MetaMorph software and the data were calculated as percentages of stained area from the total lesion area (macrophages and MMP-9) or number of cells per millimeter squared (neutrophils). Fluorescent images were obtained on a confocal microscope equipped with a digital imaging system (Carl Zeiss LSM 700, Zeiss, Baden-Württemberg, Germany) and fluorescence intensity was quantified by Image J software (NIH, Bethesda, MD, USA).