2.13 | Histology and immunohistochemistry
Aneurysms were carefully dissected and fixed in 4% paraformaldehyde overnight, followed by 30% sucrose solution for 24 hr before embedding in optimum cutting temperature compound to prepare serial frozen sections at a thickness of 7 μm. Remaining portions of the aorta were snap-frozen in liquid nitrogen and stored at −80 °C for protein analysis. Twenty serial sections of the largest aneurysm for each mouse were prepared and stained individually with hematoxylin/eosin (HE), Verhoeff-Van Gieson (VVG) and Masson’s trichrome for morphological analysis, elastin assessment and collagen deposition, respectively. The area of aneurysm was measured according to the scale with CellSens standard software. The collagen deposition was scored on a scale of 1 to 4, with 1- < 25% area in medium layer was occupied by collagen, 2–25-50% collagen deposition, 3–50-75% collagen deposition, and 4- > 75% collagen deposition. Elastin lamellae were evaluated using a grade of I to IV, with I- intact internal elastic lamina, II- mild elastin fragmentation, III- severe elastin digestion, and IV- severe elastin digestion with visible ruptured sites. The collagen deposition and elastin degradation scores among different observers blinded to the experimental groups were comparable; data are expressed as the mean value of different observers’ scores.