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. 2020 Jan 28;10:1288. doi: 10.1038/s41598-020-58339-2

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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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RANK immunostaining of CTCs ex vivo. Analysis of five rare cells and one leukocyte in a blood sample of a breast cancer patient using an Analyzer II device. Horizontally, the photos show the same cell stained for the combination (DAPI/CK FITC) of CK (green) and DAPI (violet); CK FITC only; DAPI only; CD45 APC only; and RANK PE only. The red squares indicate positively stained cells: based on RANK staining profile (sufficient signal relative to background) we classified the first two cells as RANK-positive CTCs (green squares). Based on CD45 expression, the bottom photos show a leukocyte that express RANK (light blue square), and serve as internal fluorescence control.