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. 2019 Nov 29;9:17979. doi: 10.1038/s41598-019-54262-3

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© The Author(s) 2019

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 results of immunohistochemistry and areas of endolymphatic space in the cochlear turns at E 18.5, P 6 and P 14. (A) Images after treatment at E 18.5 in the treated side of Slc26a4^−/− (c,f,i), non-treated side of Slc26a4^−/− (b,e,h), and Slc26a4^+/− control mice (a,d,g). White regions indicate the space of the endolymphatic space of the cochlea (a–c), semicircular canals (d–f), and endolymphatic sac (g–i). Blue; Hoechst. (B) The graph shows the size of the area in the scala media (SM) at each cochlear turn at E 18.5. The SM at basal and middle turn in the treated side is significantly smaller than that in the non-treated side. n = 5. *P < 0.05 (Mann-Whitney U). (C) The graph showing the relative size of the area in the endolymphatic sac (ES) to that in the non-treated control. There is no significant difference between the treated side and the non-treated side. n = 5. *P < 0.05 (Mann-Whitney U). Scale bars indicate 100 μm. (D) Images showing PENDRIN-EGFP is detectable at the stria vascularis, organ of Corti and Rosenthal’s canal in the cochlea (c,i), saccular macula (d,j), utricular macula (e,k), the ampulla of semicircular canals (f,l) in the treated Slc26a4^−/− inner ear at both P 6 and P 14. The areas of endolymphatic space in the cochlear middle turns of the treated side of Slc26a4^−/− cochlea at both P 6 and P 14 are smaller than that of the untreated side of Slc26a4^−/− cochlea. Green indicates PENDRIN-EGFP, and blue indicates Hoechst. Scale bars indicate 100 μm. (E,F) The graphs show the areas of endolymphatic space in the cochlear turns at P 6 and P 14 respectively. The areas are significantly reduced in all cochlear turns in the treated mice when compared with that in the untreated side. n = 5. *P < 0.05. **P < 0.01 (Mann-Whitney U).