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. 2018 Aug 28;8:12941. doi: 10.1038/s41598-018-31126-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Co-localization of aligned collagen fibers with THBS-2 and TNC in normal breast and IDC-s patient tissues. (a) Concentrations from quantitative LC-MS/MS analysis for collagen XII, fibronectin, thrombospondin-2, and tenascin-C. All four proteins are increased in abundance in IDC-c and IDC-s compared to normal tissue. Error bars represent sample variance, X represents the mean concentration. *p ≤ 0.05. Normal breast, n = 5; IDC-c and IDC-s, n = 4. Representative, single-plane images of collagen SHG and immunostained ECM proteins in different portions of the same patients’ tissues used for LC-MS/MS. (b) Quantifications of percent-area fluorescence within the masked-area of collagen SHG. A minimum of six ROI’s from each patient sample were used for co-localization analysis. * Indicates p < 0.05. (c) Multiphoton images of COL12A1, FN, TNC, and THBS-2 (red) with collagen SHG (green) from normal, IDC-c and IDC-s patient samples. COL12A1 significantly co-localized with IDC-c and IDC-s collagen, however it did not appear organize specifically along collagen fibers. Tenascin-C, TNC shows an increase in co-localization with only with collagen fibers in IDC-s tissues. Similarly, thrombospondin-2, THBS-2, staining is co-localized with fibrillar collagen in IDC-s samples. Scale bar = 50 µm. (d) Magnified Insets (white boxes on IDC-s TNC and THBS2 images from panel c) of straight collagen fiber (green) co-localization with TNC or THBS2 (red). Arrowheads indicate regions of co-aligned collagen and ECM protein IF.