Fluorescent microscope system

Formalin fixed, paraffin embedded sections were deparaffinized in xylenes and rehydrated through a series of graded alcohols. Tissues were processed for antigen retrieval by boiling in Diva Decloaker (pH 6.2, Biocare Medical). They were 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, Novus Biologicals, 1:100 and anti-CD68, Biorad, 1:100) or control IgG (anti-rabbit and anti-mouse 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 phosphate-buffered saline (PBS), mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Leica fluorescent microscope system. In addition, hematoxylin and eosin (H&E) stains were performed on serial sections to analyze morphology and severity of the AAA tissues. The infiltrated inflammatory cells were counted by a clinical pathologist at 3 randomly selected 20x region-of-interest of AAA and sham-control tissue slides (n=4/group). Pentachrome stain was also carried out to characterize the collagen content of AAA and sham-control tissues.

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.