Accustain elastin verhoeff s van gieson kit

The ascending aorta was dissected and fixed in 4% paraformaldehyde. The aorta was embedded in Tissue‐Tek OCT Compound Histomount (Sakura). The sample was sliced at 4‐μm cross‐sections and stained with the Accustain Elastin Verhoeff's Van Gieson kit (Sigma Aldrich), according to the manufacturer's instruction. The aorta was imaged at ×40 magnification using a Leica DM4000B microscope. For quantification, the average number of elastin breaks per elastic lamina using the whole circumference was measured by a pathologist blinded to genotype and treatment arm. Three consecutive sections from each animal were graded and the average used for that individual animal was calculated. Statistical analysis was performed using the averaged measurements from 5 different animals.

Aortic root/ascending (ASC) aortas randomly selected at 16 weeks were dissected, fixed in 4% paraformaldehyde, and embedded in Tissue‐Tek OCT Compound Histomount (Sakura, Torrance, CA). Samples were sliced at 5 μm cross‐sections and stained with Accustain Elastin Verhoeff's Van Gieson kit (Sigma Aldrich) according to the manufacturer's instructions. Three aortic sections at 50 μm increments from the most dilated portion of the ascending aorta were analyzed and imaged at ×40 magnification using a Leica DM4000B microscope. For quantification, the average number of elastin breaks per elastic lamina using the whole circumference was measured by 2 investigators blinded to genotype and treatment arm. Experiments included 5 to 7 mice per group (WT male=7; WT female=5; Fbn1 male=5; Fbn1 female=5; flutamide‐treated Fbn1 males=5; vehicle control‐treated Fbn1 males=5).