Masson's trichrome staining

Cardiac tissue samples were immobilized in 4% paraformaldehyde for 48 h at 4°C, embedded in paraffin and sliced into sections (5-µm-thick). Subsequently, sections were subjected to Masson's trichrome staining as previously described (30). The collagen fibers were blue, the muscles and elastic fibers were red, cellulose was purple-red, and the nucleus was blue-brown. Images were taken using a light microscope Nikon model with a Spot Insight camera (magnification, ×200). The fibrotic region was quantified using ImageJ software (version 1.46; National Institutes of Health). The percentage of fibrosis was calculated as fibrosis areas/total left ventricular areas ×100%.