Quantifying Cardiac Fibrosis by Trichrome Staining

Snap-frozen LV tissue was fixed in 4% PFA and embedded in paraffin. Mid-ventricular sections of 7 µm thickness were taken on Superfrost Plus glass slides (Fisher) using a Leica RM2255 microtome. These slides were then stained with Picrosirius Red, Fast Green, and Alcian Blue (RGB) trichrome stain as preciously described^{40 (link)}, to visualize collagen protein in the sections. Images were obtained using an Olympus BX51 upright microscope with a Leica DFC7000T camera and Leica Application Suite v4.6 imaging software. Areas with collagen, based on colour thresholds, were detected automatically from RGB trichrome images by a custom Fiji ImageJ 1.53c (NIH, USA) macro, which calculated percent fibrosis. The same macro was applied to all images from both genotypes at both timepoints.