Histology and Immunohistochemistry
Mouse heart tissues were formalin-fixed and paraffin-embedded for aortic valve specimens. Sections were analyzed using Haemotoxylin and Eosin (H&E) and Massonā€˜s Trichrome staining. All histological sections were semi-quantitatively graded in a blinded manner (0-3/3+ units). For immunohistochemical analyses, sections were washed in TBS/0.1% Tween-20, then blocked in 5% milk. Sections were incubated in primary antibody (1:5000) overnight, washed then incubated in biotinylated secondary antibody (1:10,000) for 30 minutes.