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. 2021 Mar 19;5:23. doi: 10.1038/s41698-021-00163-6

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© The Author(s) 2021

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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Fig. 1 — a Next-generation immunohistochemistry (NGI) workflow. An FFPE tissue section is stained, scanned and destained six times. All the scanned images are aligned, image analysis is done to obtain the data and after doing all the quality check controls, the data are analysed to obtain the final results. b Representative colour deconvoluted images of different biomarkers in the tonsil. From left to right Foxp3⁺, CD3⁺, CD8⁺, CD4⁺ and Ki67⁺. Images at 6×. c Colour overlays of different biomarkers in the tonsil (CD3⁺ in red, CD8⁺, CD4⁺ and Ki67⁺ in green from top to bottom). Images at 6×. d A representative example of all the stainings (Foxp3, CD4, KI67, CD8, CD3 and cytokeratin) in breast cancer samples and virtual image reconstruction of some of them by assigning virtual colours to the deconvoluted images. Foxp3⁺ in red, CD8⁺ in blue, CD3⁺ in green and cytokeratin in grey. The fine purple line in each image marks the tumour borders. Images at 5×.