2.6.3. Picrosirius Red Stain with Polarization Microscopy

In addition to Mason’s trichrome staining, numerous alternative methods are available for visualizing collagen in histopathological sections. In the present study, we performed picrosirius red staining with standard polarization microscopy to determine the content of type I and type III collagen in the RC tear site. In brief, hematoxylin and eosin-stained sections were covered with picrosirius red solution (PSR, Abcam, Cambridge, UK) for 60 min at 40 °C. The slide was then rinsed with acidified water, dehydrated, and cover slipped by using a toluene-based mounting medium. The PSR-stained slide was examined under an Olympus microscope (IX-71) with linear polarizers. The presence of type I and type III collagen was indicated by strong yellow-red birefringence and weak greenish birefringence, respectively. The lamp intensity was kept constant during image capture, and ImageJ software was used to quantify the collagen content in each group. Samples were evaluated at 40× magnification, and calculations were repeated in three microscopic fields.