Quantification of Aortic Collagen and Elastin

Paraffin-embedded sections (5 μm) from mice aorta were rehydrated in several changes of ethanol and Tissue-Clear^® (Sakura Finetek). Sections were then stained for collagen and elastin as previously described [13 (link)]. Sections were studied under light microscopy ZEN 2012 (Zeiss, Jena, Germany). Quantification of collagen and elastin were performed blinded. A scoring system from 1 to 4 was used with 1 defined as no degradation of elastin, 2 as low degradation, 3 as intermediate degradation, and 4 as high degradation. For collagen quantification, 1 defined as low amount of collagen in all layers of the aorta, 2 as low amount of collagen in aorta media or in aorta adventitia, 3 as high amount of collagen in aorta media or in aorta adventitia, and 4 as high amount of collagen in all layers of the aorta. The suprarenal aortic diameter was measured as the adventitial diameter and the definition of an aneurysm was set as a 1.5-fold enlargement of leading edge to leading edge. Measurements of the aortic diameter was performed using ZEN 2012 (blue edition, Carl Zeiss Microscopy GmbH, 2011 ver 1.1.2.0, Oberkochen, Germany). To measure aortic outer diameter, the largest dilated part of the aorta was taken for this purpose.