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. 2017 Nov 14;7:15580. doi: 10.1038/s41598-017-15798-4

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© The Author(s) 2017

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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Multiplexed immunohistochemistry for immune cells in prostate tumour (patient 1). (a) Fluorescence (FL) and brightfield (CHR) images are acquired and (b) registered using nuclei information from both images (Hoechst and haematoxylin). (c) Cell segmentation and classification is based on nuclei, epithelium marker expression (Pan-Epi = Pan-CK + ECad), and cell-type specific marker expression (CD45 for leukocytes). Scale bar 500 µm. CHR, chromogenic; FL, fluorescence; IHC, immunohistochemistry; Pan-CK, pan-cytokeratin; Pan-Epi; pan-epithelium.