Anti mouse and anti rabbit igg

After directly placing in saline upon excision of the AAA during surgery, human specimens were fixed in 10% formalin, embedded in OCT compound, frozen, and sectioned. Frozen sections were hydrated in PBS, and blocked in 10% donkey serum for 1 h to prevent nonspecific binding. The sections were then incubated overnight at 4°C in primary antibody (anti-CCR2, Abcam, 1:400 and anti-CD68, Millipore Sigma, 1:100) or control IgG (anti-mouse and anti-rabbit IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 1 h at room temperature, and sections were washed in PBS, mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Leica fluorescent microscope system. In addition, hematoxylin and eosin (H&E) and Verhoeff-Van Gieson (VVG) stains were performed on serial sections to analyze morphology and severity of the AAA tissues.

After harvest, whole rat aortas were washed in PBS and then fixed in 30% sucrose mixed with 4% PFA for 2–3 h. Tissues were then blocked in 1% BSA with 1% Triton X-100 for 4 h to prevent nonspecific binding, and whole tissues were then incubated for 48 h at 4°C in primary antibody (anti-CCR2, Novus Bio, 1:50 and anti-CD68, Bio-Rad, 1:100) or control IgG (anti-mouse and anti-rabbit IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 24 h at 4°C, and tissues were washed in PBS and incubated with DAPI (1:700 in PBS from Sigma). Tissues were then cleared in 70% ethanol, followed by 100% ethanol overnight. Whole tissues were then mounted in aqueous mounting media and imaged using a Zeiss LSM 880 II Airyscan FAST confocal microscope.