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. 2019 Sep 2;9:12645. doi: 10.1038/s41598-019-49164-3

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© The Author(s) 2019

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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Comparison of multi-staining protocol with FACS and standard IHC techniques. (A) Representative images of WT and E1 TG DSS-treated colons multiple stained for CD3 (red), CD4 (green), CD8a (yellow) and CD45R (magenta). Original magnification, 200x. (B) Leukocyte analysis of the LP (a and b) and EP (c and d) fractions performed with mIHC, calculating the percentage of each population on the number of total cells (on the left); leukocyte analysis of the LP (e and f) and EP (g and h) fractions performed with FACS, recalculating the percentage of each population on the total events (on the right). (C) Comparison between mIHC with standard IHC results. We converted the number of positive cells coming from inForm software analysis into the same score applied in the histology evaluation as reported in Fig. 2. Bar graphs show mean values ± s.d. from at least three mice individually tested per group.