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.