Quantification of Atrial Fibrosis in Mice

Quantification of atrial interstitial fibrosis was performed using the Picrosirius red staining technique, as previously described [59 (link)]. The left atria were removed from the mouse hearts under anesthesia with 2% isoflurane, and fixed overnight with 10% buffered formalin at 4 °C. The atria were embedded in paraffin and cut to a thickness of 8 µm using a microtome. Sections were deparaffined using xylene and rehydrated with an ethanol-water gradient. Sections were then stained for 1 h with a saturated picric acid solution containing 0.1% Direct Red 80 (Sigma-Aldrich Corp., St. Louis, MO, USA), and washed twice with 0.5% acetic acid solution. The slides were dehydrated with a water-ethanol gradient before cover slipping them with Permount medium (Fisher Scientific, St-Laurent, Qc, Canada). Images were acquired using Qicam optic camera (Qimaging Corp., Tucson, AZ, USA) mounted on a brightfield microscope and analyzed using ImageJ software, version 1.8.0 (National Institutes of Health [NIH], Bethesda, MD, USA).