Visualization of collagen fibers via picrosirius red staining and quantification of collagen content.

<p>Picrosirius red staining of <b>(A)</b> healthy and <b>(B)</b> diseased leaflets was visualized using brightfield microscopy and polarized light. Birefringence hue and amount were quantified as a percent of total tissue area (N = 5, n = 3) in the <b>(C)</b> fibrosa and <b>(D)</b> spongiosa. Collagen production was also quantified via <b>(E)</b> qRT-PCR analysis of <i>COL1A1</i> gene expression (N = 4, n = 3) and <b>(F)</b> measurement of total collagen protein via dot blot (N = 5 healthy; N = 4 diseased, n = 3).