In immunohistochemical/immunocytochemical staining, the positivity was evaluated by image analysis performed using the Leica Q500 MC Image Analysis System (Leica, Cambridge, UK) as previously described [18 (link)]. For picrosirius red staining, a total of 10 fields were randomly chosen per mouse and images were viewed with brightfield illumination as well as with polarization contrast illumination at 40x. Picrosirius red is a birefringent molecule that binds to collagens. The complex fibrillar collagen/sirius red can be detected under polarized light, and the collagen bundles are red, yellow, and green. Collagen expression was quantified under brightfield illumination.
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