Quantitative Analysis of Collagen Birefringence

Picrosirius red staining was performed and analyzed as previously described [23 (link)]. Briefly, tissues were deparaffinized, hydrated and stained with 0.1% Sirius red F3BA in saturated aqueous picric acid (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Nuclei were counterstained with Weigert’s hematoxylin (Sigma-Aldrich, St. Louis, MO, USA), dehydrated, and mounted. Birefringence was captured under circular polarized light and the density of 4 different colors (green, yellow, orange, red, indicative of elevating collagen bundle thickness) was quantified. The ratio of individual colors was expressed relative to the number of total positive pixels. Total collagen was calculated as the proportion of positive pixels relative to the tissue area calculated in ImageJ with thresholding of a bright field image captured at the same position as with the polarized light.