Picrosirius red staining

The heart was removed and fixed in 10% neutral buffered formalin overnight at 4 °C. The heart tissue was dehydrated in a gradient of ethanol concentrations of 70%, 80%, 95%, and 100%, followed by incubation in xylene for 15 min and embedding in paraffin blocks at 60 °C for 1 h. Thin (5 μm) myocardial sections were cut using a microtome (Leica), placed onto a Super frost microscopic slides (VWR, Cat# 48311–703), and left at 37 °C overnight to dry.

To detect and quantify myocardial fibrosis, thin myocardial sections were deparaffinized with xylene for 15 min twice at room temperature and were rehydrated in a series of ethanol concentrations of 100%, 95%, 80%, 70%, and 50% for 2 min each at room temperature. The sections were stained with picrosirius red according to the manufacturer’s protocol (Sigma Aldrich, Cat# P6744–1GA), mounted in a xylene based mounting medium, and were imaged using an Olympus microscope equipped with a digital camera (Olympus BX40). Collagen volume fraction (CVF) was calculated in 15–20 high microscopic fields (×20) per section, 6 sections per heart, and in a minimum of 5 mice per group using Image J software, as published^[30,36].