AFOG staining and quantification of collagen deposition

AFOG staining was performed on 10 μm sections as described⁵. Samples were first incubated at 60°C for 2 hr with Bouin preheated at 60°C for 30 min; slides were then rinsed in running water for 30 min, and incubated with phosphomolybdic acid solution, followed by the sample incubation in AFGO staining solution for 10 min. For quantification of collagen deposition, collagen⁺ area was manually measured using ImageJ and normalized by the ventricular size and normalized by the entire ventricle size. Two to four largest sections were analyzed for each heart.