Pa5 13199

Histology of the aortic root and the mid-ascending aorta for LDLR^-/-Apobec1^-/- mice was performed 21 days after MI or sham procedure. Blood was removed from the arterial system by infusion of isothermic buffered saline after which the aorta was perfusion-fixed. Trans-axial sections of the aortic root at the level of the sinuses were stained with Masson’s trichrome to assess the plaque area within the internal elastic lamina, and collagen content. Immunofluorescence histology was performed with anti-mouse primary mAb against Mac-2 (M3/38, Invitrogen, Waltham, Massachusetts, USA) for monocytes/macrophages, against CD41 (ab181582, Abcam, Cambridge, United Kingdom) for platelets, and against matrix metalloproteinase-9 (MMP-9) (PA5-13199, ThermoFisher). Staining for evidence of vascular cell signaling through platelet-derived TGFβ1 was performed with mAb against β-catenin (51067-2-AP, Proteintech, Rosemount, IL) and phosphorylated SMAD2 (pSMAD2) (H.205.4, Invitrogen). Secondary staining was performed with species-appropriate secondary polyclonal antibodies labeled with ALEXA fluorochromes (Fluor-488, Fluor-568, or Fluor-647) and observations were made by confocal fluorescent microscopy (TCS SP5, Leica Microsystems, Buffalo Grove, IL). Spatial extent of plaque size, collagen content, or fluorescent staining area were quantified using Image-J.

Immunohistochemical assays were performed with the following primary antibodies: rat F4/80 antibody (Cl-A3-1) NB600-404 (1:50 dilutions; Novus, Centennial, Colorado, USA); rabbit matrix metalloproteinase (MMP) 9 antibody PA5-13199 (1:50 dilution; Thermo Scientific, Waltham, MA, USA); rabbit MMP2 antibody PA1-16667 (4 μg/mL dilution; Thermo Scientific, Waltham, MA, USA); and goat CCL4 antibody (M20) sc-1387 (1:50 dilution; Santa Cruz Biotechnologies, Dallas, TX, USA). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA. The reaction was visualized by staining with 3,3′-diaminobenzidine (DAB) or fluorescence.