4.2. Tissue Collection, Lesion Analysis, and Immunohistochemistry

At the end of WD treatment, mice were euthanized with CO₂. Blood was collected by cardiac puncture, and vascular perfusion was performed with sterile RNase-free PBS. After perfusion, heart and the aortic arch were dissected and preserved for lesion and immunohistochemistry analyses. En face lipid accumulation was measured in the aortic arch using Sudan IV staining. Plaque area was calculated as the sum of total plaque area in the aortic arch (excluding branching vessels). Aortic root lesions were evaluated in hearts from male transplanted mice that were serially sectioned from the proximal 1 mm of the aortic root on a cryostat. Lesion size was determined from 8 hematoxylin- and Oil Red O-stained sections, collected every 100 μm over a 1 mm segment of the aortic root. Lesions were quantified as previously described [20].

Primary rat anti-mouse antibodies to CD68 (AbD Serotec, Oxford, UK), CD3 (Southern Biotech, Birmingham, AL, USA), VCAM-1 (BD Biosciences, Franklin Lakes, NJ, USA), and rabbit anti-mouse antibody to alpha smooth muscle cell actin (Abcam, Cambridge, UK) were applied to acetone-fixed cryosections, followed by biotinylated rabbit anti-rat or goat anti-rabbit IgG. Mouse anti-mouse IA^b (BD Biosciences, Franklin Lakes, NJ, USA) was conjugated directly to biotin. Staining reactions developed using the VECTASTAIN ABC kit and diaminobenzidine (Vector Laboratories, Burlingame, CA, USA). Immunohistochemical data was obtained using Qwin computerized analysis (Leica, Wetzlar, Germany) of stained sections. Samples that were compromised during processing or analysis were excluded from the study. For the assessment of plaques, samples were coded, and the evaluation performed by trained persons, which were blinded to the treatment groups.