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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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The FACT protocol is compatible with immunohistochemistry and performs well in both antibody-based and transgene-based imaging. (A and B) Three-dimensional 200-µm-thick volumes of cerebral cortex after antibody staining and YFP-labeled microglia in transgenic mice. (C and D) Three dimensional reconstructions of microglia surface area and volume by the Imaris Surface algorithm and color sorting of the areas by Imaris Vantage from white (highest area) to purple (lowest area). Higher detection of microglia signal was achieved in the Iba1 immunostaining compared to transgene-YFP-microglia . (E and F) Comparisons of corrected microglia numbers and corrected mean signal intensities between the two FACT-based detection protocols after semiautomatic counting with the Imaris Surface algorithm. No significant differences were evident (p > 0.05). (G and H) Comparisons of microglial cell surface area and microglial cell volume between the two FACT-based detection protocols after semiautomatic measurement with the Imaris Surface algorithm. Greater signal detection was seen in the antibody-labeledmicroglia compared to the transgene-labeled microglia (p < 0.05). Scale bars are 100 μm.