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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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Compatibility of RTF with virus labeling, immunostaining and DiI-labeling. (a) The injection site of rabies virus (red point) and position of brain section (cyan line) are shown in the schematic diagram. This diagram was drawn by referring to The Mouse Brain in Stereotaxic Coordinates, 2nd edition, Franklin, K.B.J. and Paxinos, G. After transducing with RV-ΔG-dsRed, the brain sections at indicated positions were dissected and imaged before and after clearing with RTF. The maximum intensity projections of enlarged region are indicated with white box in the image of half brain section. DsRed in red and EGFP in green. The images showed that endogenous GFP and virally delivered dsRed expression both presented fine fluorescent signal. Scale bar, 500 μm for first and third panel; 50 μm for second panel; 100 μm for fourth panel. (b) Cx3cr1-GFP hippocampus sections immunolabeled with microglial cells marker anti-GFP, cleared with RTF; The GFP and anti-GFP fluorescence (with a secondary antibody conjugated to Alexa Fluor 594) were visualized. Scale bar, 100 μm. (c) Merged stack of DiI-labeled cells in MD region imaged from 400 μm brain section, before and after clearing. Scale bar, 20 μm.