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. 2020 Jul 16;10:11715. doi: 10.1038/s41598-020-68638-3

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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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Immunostaining for NKCC1 in astrocytes and microglial cells. Micrographs of 1 µm thick laser scanning confocal optical sections illustrating the colocalization between immunolabeling for NKCC1 (red; b, e) and immunoreactivity for markers that are specific to astrocytes (GFAP, green; a) and microglial cells (IBA1, green; d) in the superficial spinal dorsal horn. Mixed colors (yellow) on the superimposed images (c, f) indicate double-labeled structures. Micrographs of short series of confocal optical sections double immunostained for NKCC1 (red) and GFAP (green) or IBA1 (green) show the colocalization between NKCC1 and GFAP (g) as well as IBA1 (h) illustrated in X–Y, X–Z and Y–Z projections. Two selected points (labeled with 1 and 2) of colocalization between the markers are at the crossing point of two lines indicating the planes through which orthogonal views of X–Z and Y–Z projections were drawn. Small inserts in the upper right corner of both g and h show the selected two points in X–Y dimension without the lines. The corresponding X–Z and Y–Z projections are beside and below these inserts. According to the orthogonal images, as it is indicated by the mixed color (yellow), NKCC1 immunostained puncta 1 and 2 are within the confines of the GFAP as well as the IBA1 immunoreactive profiles. Double stained spots are marked with arrows. Scale bar: 5 µm (a–f), 2 µm (g, h).