Masson's trichrome staining

According to a previous report (16), Masson's trichrome staining was conducted to evaluate the extent of atrial fibrosis. After the rats were sacrificed, atrium samples were rapidly excised, rinsed with PBS, fixed in 4% paraformaldehyde at 4°C overnight, and embedded in paraffin. Then, 5-µm-thick sections at of the atrium were cut. Masson's trichrome kit (cat. no. HT15; Sigma-Aldrich; Merck KGaA) was used according to the manufacturer's instruction. ImageJ software (Version 1.8; National Institutes of Health) was used to evaluate the area of fibrosis. Each section was examined in 5 randomly-selected high-power fields (×400) and middle-power fields (×200) under a light microscope. Collagen volume fraction (CVF)=Collagen area (blue)/Total area ×100%.