Quantifying Collagen Content in Vascular Tissue

To compare the difference in vascular collagen content between groups, the tissue sections were stained with Sirius Red. Sections were deparaffinized and hydrated to distilled water. Then, sections were placed in Sirius Red solution for 8 min followed by dehydration and mounting. Sirius Red dye is strongly acidic and easy to combine with basic groups in collagen molecules. Collagen fibers were stained a red–orange color and observed by an image acquisition system (Aerio CS2, Leica, Germany). Image Pro Plus version 7.0 was used to calculate the percentage of collagen fibers in tunica media. Sirius Red bonds with collagen fibers, which makes collagen fibers produce obvious birefringence. Under the polarizing microscope (DM2700P, Leica, Germany; polarization angle is 90 degrees), through the characteristics of enhanced birefringence light, type I and III collagen fibers show different colors, and type I collagen fibers show red–orange red; type III collagen fibers are green.
Autofluorescence of elastic fibers in tissue sections stained H&E was recorded with excitation wavelength at 460 nm–550 nm (Olympus 1 × 2 UCB) [32 (link),82 (link)].