2.9. Sirius Red Staining

4–6 μm thick sections were deparaffinized and rehydrated using xylene, 100% ethanol, 95% ethanol, and 70% ethanol for 3 min each and stained using Picro-Sirius Red staining kit (Abcam, Cambridge, MA). Picro-Sirius Red solution was applied to completely cover the tissue section and incubated for 1 hour and then rinsed twice in acetic acid solution and absolute alcohol. Sections were then dehydrated by dipping twice in absolute alcohol and then mounted with Eukitt quick hardening mounting medium (Sigma-Aldrich, St. Louis, MO). Picro-Sirius Red stains collagen 1 and collagen 3 fibers in the tissue section. Staining of collagen fibers was assessed based on five 20x magnification fields per animal per group in a blinded fashion using Zeiss 510 microscope (Carl Zeiss, Thornwood, NY). Percent of fibrosis was calculated based on the intensity of Sirius Red staining using ImageJ (NIH, Bethesda, MD).