Quantifying Myocardial Fibrosis by Collagen Staining

Collagen staining was done as described^{15 (link)}. In brief, the paraffin embedded sections were dewaxed using histoclear (10 min X 2). Multiple ethanol washes were performed in series followed by 1-hour incubation with picrosirius red solution (Sigma). The sections were washed with acetic acid for 45 min followed by 100% ethanol for 1 min, and finally mounted with resinous medium (Cytoseal). Images were taken using a slide scanner - Hamamatsu Nanozoomer 2.0HT. Percent fibrosis of the LV was quantified using Aperio Imagescope v12.1.0.5029 software. The LV wall was traced, excluding the pericardium and a line was drawn bisecting the muscular septum equally to calculate the LV region. The percent fibrosis was quantified using strong positive signal divided by total signal.