Polarized light imaging of Picrosirius red staining

Polarized light microscopy was performed on fixed, deparaffinized, and rehydrated 4-μm sections stained with 0.1% Picrosirius red (Polysciences, 29401-250). Polarized light signal of fibrillar collagen was taken using an Olympus U-Pot polarizer and an Olympus U-ANT transmitted light analyzer fitted to a DM4000 microscope (Leica). Quantification of birefringent signal was analyzed using ImageJ. Briefly, hue-saturation balance thresholding was applied (high birefringence/red-orange 0>H<29 | 0>S<255 | 70>B<255, medium birefringence/yellow 30>H<44 | 0>S<255 | 70>B<255, and low birefringence/green 45>H<245 | 0>S<255 | 70>B<255). Relative area of fibers was then calculated as percentage of total fibers (0>H<245 | 0>S<255 | 70>B<255).