Anti mouse ly6g

After shaving the furs and sterilizing the skin with 70% ethanol, mice were administered with
lipofectamine-packed miR-133a-3p (20 μg/mouse) or lipofectamine alone through intra-peritoneal injection (i.p.) using
31 G insulin syringe. The injection site was subsequently covered by an adhesive 3M Tegaderm film to prevent infection. Twenty
hours later, the peritoneal lavage was harvested as described previously (21 (link)). In
brief, 2 ml of normal saline was injected into the peritoneal space and mixed thoroughly by gentle massage of the abdomen.
About 1.2 ml of the peritoneal lavage was collected and centrifuged. The cell pellets were suspended. Total peritoneal cells
were manually counted. Eight × 10⁵ cells were incubated with 1:100 diluted specific anti-mouse Ly-6G (BD
Biosciences, San Jose, CA) and anti-mouse F4/80 (eBiosciences, San Diego, CA) at 4°C for 30 min. After washing, the
neutrophils, resident macrophages, and recruited monocytes were determined by flow cytometry gating as
Ly-6G⁺, F4/80^high, or F4/80^low, respectively.

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).