M2 macrophages arginine type 1

Aortic tissue was fixed in 4% buffered formaldehyde for 24 hours, transferred to 70% ethanol, and then embedded in paraffin. Cross-sectional samples were prepared using Verhoeff-van Gieson trichrome to stain for collagen and elastin. Two independent blinded reviewers scored elastin breaks using a previously published scale.^{9 (link)} Immunohistochemical staining was also performed for anti-mouse α smooth muscle actin (α-SMA) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), macrophages (anti-rat Mac-2; 1:10,000, Cedarlane Laboratories, Burlington, ON, Canada), macrophage-specific (Galectin-3 ligand; 1:200, Abcam, and CD68 ligand; 1:200, Abcam), M1 macrophages (IL-1β ligand; 1:200, Abcam), M2 macrophages (Arginine type 1; 1:200; Abcam), and CD3 for T cells (1:500; Santa CruzBiotechnology). Visualization color development was completed using alkaline phosphatase (Dako, Glostrup, Denmark) for α-SMA, and diaminobenzidine for Mac-2, anti-neutrophil, and CD3. ·For elastin grading, 1 corresponded to no degradation, 2= mild degradation, 3 =moderate degradation, and 4= severe degradation. For smooth muscle cell (SMC) grading, 1 corresponded to no SMC loss, 2 = mild SMC loss, 3 = moderate SMC loss, and 4 = severe SMC loss.

Cross-section samples were prepared using Verhoeff-Van Gieson (VVG) Trichrome to stain for collagen and elastin. Two independent blinded reviewers scored elastin breaks using the following scale: 1=concentric heavy rings of elastin, 2=mild elastolysis, 3=moderate elastolysis, and 4=extensive elastolysis. These scores were averaged and compared. Immunohistochemical staining was also performed for anti-mouse α smooth muscle actin (α-SMA) (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), macrophages (anti-rat Mac-2; 1:10,000, Cedarlane Laboratories, Burlington, ON, Canada), M1 macrophages (IL-1β ligand; 1:200, Abcam), M2 macrophages (Arginine type 1; 1:200; Abcam), and CD3 for T cells (1:500; Santa Cruz Biotechnology). Visualization color development was completed using alkaline phosphatase (Dako, Glostrup, Denmark) for α-SMA, and diaminobenzidine for Mac-2, anti-neutrophil, and CD3.
Images of the sectioned aortas were acquired and analyzed as previously published.^{10 (link)} In short, images are obtained using AxioCam 4.6 software with 4x objective and an AxioCam MRc camera (Carl Zeiss GmBh). For quantification, the integrated optical density of the positive staining area in the cross sectional image of the aortic tissue sample was selected and measured using Image-Pro Plus 7.0 (Media Cybernetics, Bethesda, MD, USA).