Quantification of dermal collagen density in wild-type and Cav-1-/- mice. (A) Electron microscopy (EM) images of dermal collagen from wild-type and Cav-1-/- mice were analyzed with a custom Image J algorithm (Sup. Fig. 1). Using this approach, we determined the collagen density, as number of fibers per unit area. We observed that Cav-1-/- mice contain a significantly larger number of dermal collagen fibrils per unit area, than wild-type animals. (***p < 0.001, as determined by Mann-Whitney Rank Sum Test using SigmaPlot). (B–E) Picrosirius red staining of skin from wild-type and Cav-1-/- mice. Polarized light images of the dermis and the deeper dermis around skin appendages and blood vessels of wild-type (B and D) and Cav-1-/- mice (C and E) were stained with picrosirius red. Mature collagen fibers stained with picrosirius dye appear red when observed under polarized light, whereas less mature fibers, with fewer cross-links, appear yellow and green. Note that although both groups display mature collagen fibers in the dermis, a larger amount of collagen accumulation was found in the dermis, the deeper dermis, and around blood vessels of Cav-1-/- skin, as compared to wild-type mice. Scale bar = 200 µm.