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 Massonās 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.