I would like to point out a discrepancy in the claims of this protocol. In the last paragraph of the Background section of this protocol, the authors remark: "The amount of polarized light absorbed by the Sirius red dye stringently depends on the orientation of the collagen bundles ENABLING to differentiate different collagen types (Junqueira et al., 1979; Lattouf et al., 2014)." This claim is in stark contrast with the cited publication Lattouf at al., 2014. Already the abstract of this publication concludes: "Using a simple histological example, our study illustrates the INABILITY of Picrosirius red staining to differentiate collagen types, ..." I declare that I am no expert in collagen or polarization microscopy. Yet, it is not surprising to me that polarization microscopy cannot reveal they collagen type based on the color. The color is result of light interference due to the birefringence. The optical path difference introduced through birefringence, and thus the apparent color, will depend both on the optical properties of the stained collagen, its amount in the optical path through the specimen and its orientation in respect to the polarization of the illuminating light. In a heterogeneous structure like a tissue section, the amount of stained collagen in the optical path cannot be separated from the birefringence of the different stained collagen types by virtue of polarization microscopy. Therefore it should not be surprising that Lattouff et al. 2014 found experimentally that apparent color in the polarization microscopy images of the Picrosirius red-stained collagen do not infer the type of collagen in the specimen. They have done a simple yet robust experiment to disprove this hypothesis. They prepared a sample of collagenated tissue stained with Picrosirius red and observed it in a polarization microscope. They seen different colors throughout the sample. They then rotated the sample by 90 degrees and they found that various patches of collagen in the sample changed their color as they rotated the sample. Clearly, the color depends on the orientation of the sample in the polarized light as well as the properties and amount of stained collagen in the specimen. If the orientation of mounting of the specimen changes the color, the color on its own cannot be used to infer the collagen type. I would like to read a comment from the authors of the protocol on my critique. 11/20/2018 3:00:00 AM Reply