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. 2017 Aug 29;7:9895. doi: 10.1038/s41598-017-10204-5

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© The Author(s) 2017

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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Comparisons of 400-µm-thick volumes of Z-stack images of microglia after testing seven protocols. (A–G) Three-dimensional blocks of microglia populations of the cerebral cortex in transgenic mice after clearing with the various protocols. The greatest signal and maximal architectural details were achieved with the FACT protocol. For all images, confocal microscopy was employed at 25× magnification. (H) Comparisons of corrected microglia numbers between the different clearing protocols (after semiautomatic counting with the Imaris Surface algorithm) reveal a lack of significant differences between the groups (p > 0.05). (I) Comparisons of the corrected mean intensities of the signals between the different protocols. The highest signal detection was achieved with the FACT protocol. Scale bars are 100 μm.