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. 2019 Oct 1;9:14063. doi: 10.1038/s41598-019-50506-4

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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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Immunofluorescence and Western blot analysis of ionized calcium binding adaptor molecule 1 (Iba-1), cluster of differentiation 86 (CD86), and inducible nitric oxide synthase (iNOS) counterstained with 4′-6-diamino-2-phenylindole (DAPI) in transverse sections of rat spinal cord treated with phosphate-buffered saline (PBS) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) 24 h after injury. Representative images from the ventral region of (a) PBS- and (b) DIDS-treated groups stained with Iba-1 (green) and counterstained with DAPI (blue), with high magnification view of the respective selected area and tagged skeleton showing its voxels. (c) Graph indicating the average branch length of Iba-1-positive cells in PBS- and DIDS-treated groups. Representative images from the ventral region of (d) PBS- and (e) DIDS-treated groups stained with CD86 (green) and counterstained with DAPI (blue), with high magnification view of the respective selected area. (f) Graph indicating the number of CD86-positive cells in PBS- and DIDS-treated groups. Representative images from the ventral region of (g) PBS- and (h) DIDS-treated group stained with iNOS (green) and counterstained with DAPI (blue), with high magnification view of the respective selected area. (i) Graph indicating the number of iNOS-positive cells in PBS- and DIDS-treated groups. (j) Representative iNOS and amido black (AB) bands of spinal cord samples from PBS- and DIDS-treated animals. Quantification of the optical density of iNOS bands, comparing PBS- and DIDS-treated animals. Bars represent standard error of the mean. Scale bar: 50 μm.