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. 2017 Sep 26;7:12297. doi: 10.1038/s41598-017-12069-0

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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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Accumulation of autophagy reporter substrates in the cerebellum of murine models of RTT. Immunohistochemistry analysis of the cerebellum from 9-weeks-old wild-type mice and 5- (asymptomatic) and 9-weeks (symptomatic) old RTT mice knock-out for MeCP2 (n = 3 for each experimental group). For the three experimental conditions, the organs isolated from the different animals were studied. Slices (n = 4) from each organ were probed with anti-p62/SQSTM1 (a) and an anti-Ub antibody (b). An age-linear increase in the staining for both p62/SQSTM1 and Ub was observed in all the cerebellum layers of RTT mice compared to the cerebellum of wild-type animals. (c) Bar graph showing the semiquantitative evaluation of p62/SQSTM1 and Ub immunoreactivity, expressed as arbitrary unit. The results were expressed as mean number ± S.E., with an interobserver reproducibility of ±95%. The staining of the cerebellum of 5-weeks mice was compared to that of wild-type mice, and the staining of the 9-weeks mice to that of 5-weeks mice. Differences were evaluated by a Student’s τ test and were considered significant at p value ≤ 0.05.