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. 2020 Nov 18;10:20059. doi: 10.1038/s41598-020-77036-8

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Immunohistochemical staining (IHC) and measurement of CD8⁺ tumor-infiltrating lymphocytes (TILs). (a) Low magnification image with an indication of tumor area by a solid green line. Areas of cystic space, non-specific stain, and dust were excluded from analysis (scale bar = 1 mm). (b) Converted image of 1A for the analysis scanned by Aperio (Leica Biosystems). (c) Representative image of high magnification. Many lymphocytes within the tumor area were stained with IHC staining for CD8 (scale bar = 50 μm). (d) Representative Aperio image of 1C. Lymphocytes are marked with blue (negative), yellow (1 +), orange (2 +), or brown (3 +) depending on staining intensity.