Tissue samples were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin for evaluation of tissue architecture. The slides were washed with tap water and stained a with 0.1% fast green FCF for 10 min, followed by washing with acetic acid and staining with picrosirius red F3BA for 1 h. The slides were then washed with acidified water, dehydrated, cleared, and mounted in resinous medium. Sirius red/fast green (SR/FG) stain was chosen for its ability to differentiate type I from type III collagen. Sirius red is a strong anionic dye that stains collagen by reacting, via its sulfonic acid groups, with basic groups present in the collagen molecule. The dye molecules attach to the collagen fibers in such a way that their long axes are parallel. This configuration between the dye and the collagen results in an enhanced birefringency [14 (link)]. When viewed under cross-polarized light, the collagen fibers appear distinctly different from one another, with the type I collagen fibers appearing bright yellow-red/orange, while the type III collagen fibers appear green-blue. Slides were examined using cross-polarized light microscopy with an Axioskop 40 microscope (Carl Zeiss, Thornwood, NY) equipped with a Zeiss Axiocam at 400× magnification. A total of ten high-resolution images were captured of each slide, taking care not to overlap the images, ensuring ample representation of the field. The images were stored as multidimensional ZVI files for analysis.