The aortas were prepared en face and stained Oil Red O (Sigma-Aldrich, St. Louis, MO, USA). The aortic lesion area and total aortic area were calculated using LSM Image Browser software.
The hearts with the ascending aorta were embedded in OCT compound (CellPath, Newtown, UK), snap frozen and sectioned (10 μm thickness) for histological and immunohistochemical analysis, according to the standardized cross-section protocol, as described before [51 (link),52 (link)]. To evaluate the lesion area and plaque collagen content, nine sections per animal were stained Oil Red O (Sigma-Aldrich, St. Louis, MO, USA). Immunohistochemistry was performed with antibodies against CD68 (dilution 1:800; Serotec, Kidlington, UK) and smooth muscle α-actin (SMA) (dilution 1:800; Sigma-Aldrich, St. Louis, MO, USA). In situ zymography was performed to demonstrate non-specific activity of gelatinases using the standard protocol [53 (link)]. All section images were captured using an Olympus Camedia DP71 digital camera and analyzed using LSM Image Browser software (Zeiss, Jena, Germany).
The hearts with the ascending aorta were embedded in OCT compound (CellPath, Newtown, UK), snap frozen and sectioned (10 μm thickness) for histological and immunohistochemical analysis, according to the standardized cross-section protocol, as described before [51 (link),52 (link)]. To evaluate the lesion area and plaque collagen content, nine sections per animal were stained Oil Red O (Sigma-Aldrich, St. Louis, MO, USA). Immunohistochemistry was performed with antibodies against CD68 (dilution 1:800; Serotec, Kidlington, UK) and smooth muscle α-actin (SMA) (dilution 1:800; Sigma-Aldrich, St. Louis, MO, USA). In situ zymography was performed to demonstrate non-specific activity of gelatinases using the standard protocol [53 (link)]. All section images were captured using an Olympus Camedia DP71 digital camera and analyzed using LSM Image Browser software (Zeiss, Jena, Germany).