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. 2020 Sep 24;15:116. doi: 10.1186/s13000-020-01021-y

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© The Author(s) 2020

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — IHC markers in the PDAC mIHC panel to study tumor immune interactions. a Traditional IHC to stain six adjacent serial tissue sections with one biomarker per tissue section. Each biomarker is designated by a specific color. b Representative images from one marker IHC in serial tissue sections showing expression in the same region of interest (except for examples of sparsely distributed B-cells from a different area in the corresponding histologic section). Each inset shows the cellular expression of the corresponding marker at higher magnification. C. Multiplex IHC (mIHC) in adjacent serial section. d Hematoxylin and eosin (H&E) in adjacent serial sections with delineation of the tumor region by a pathologist. e mIHC with six IHC markers on a single tissue section with PDAC. f At the highest magnification, each of the five immune cell classes are labeled with a white arrowhead outlined in the color corresponding to chromophore color. T-cell subtypes include CD3+ (yellow), CD4+ (teal), CD8+ (purple); B-cells denoted by CD20+ (red); and myeloid cells identified by CD16+ (silver-black). g Indirect mIHC using hapten-conjugated secondary antibodies. Primary antibody binds to target antigen; secondary anti-mouse or anti-rabbit IgG antibody conjugated to synthetic haptens (HQ or NP) to bind primary antibody; and anti-hapten tertiary antibody conjugated to multiple enzyme molecules (e.g. horseradish peroxidase (HRP) or alkaline phosphatase (AP)) to bind secondary antibody. Chromogen substrate reacts with enzymes to generate insoluble unique color signal at the site of the targeted antigen