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. 2018 Jan 31;8:1964. doi: 10.1038/s41598-018-20306-3

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© The Author(s) 2018

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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Preservation of GFP fluorescence and morphology maintanence. (a) Thy1-GFP-M line adult brain slices (1-mm-thick) were prepared and imaged before and after clearing with RTF and Clear^T2. Maximum intensity projections of representative images were shown. Scale bar, 100 μm. (b) Quantifications of fluorescence intensity for GFP. The relative total fluorescence intensity after RTF clearing showed significantly higher value than Clear^T2. Data are presented as mean ± s.d (n = 5). **P < 0.01 (Mann-Whitney U test). (c) Orthogonal view (x-z) of image stacks obtained from Cx3cr1-GFP mouse brain slices (1-mm-thick). Scale bar, 50 μm. The insets were the optical slices (x-y view) indicated with dashed red line in C. (d) Represented curves of image contrast against z depth. (e) Bar plot of imaging depth calculated based on contrast decay for PBS, Clear^T2 and RTF. Data are presented as mean ± s.d (n = 4). ***P < 0.001 (One-way ANOVA followed by Dunnett’s post hoc test). (f) Typical pyramidal neurons imaged before and after clearing. Scale bar, 20 μm in top, 5 μm in bottom. (g) Reconstruction of neurons in hippocampus of Thy1-GFP-M mouse. Scale bar, 1000 μm in left, 50 μm in right.