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. 2020 Sep 1;10:14408. doi: 10.1038/s41598-020-71347-6

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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 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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Automated tissue image acquisition using HCS. (a) A whole frozen colon tissue slice from a αCDH-A647 treated animal was mounted to a standard microscope slide and stained with anti-EpCAM and DAPI. (b) A slide holder with standard microtiter plate footprint was built to house 4 tissue slides in order to facilitate automated image acquisition using the PHENIX HCS reader. (c) A 5 × air objective lens was applied to localize all nuclei (blue), thus defining ROI and providing an optimal Z height starting point. Side images are orthogonal views of maximum nuclear intensity at different Z heights. (d) Automated multi-color (blue: nucleus; green: EpCAM; red: αCDH-A647) rescan on ROI guided by PRECISCAN was conducted using a 20 × water-immersion objective lens. Images from all FOVs were then montaged for analysis. Yellow arrow points to one example of an area where αCDH-A647 was trapped in the interstitial space. (e) Different features of nuclei (top) and epithelia (bottom).