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. 2018 Apr 26;8:6610. doi: 10.1038/s41598-018-24899-7

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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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Physiological state of [Ca²⁺]_i in the epidermis. Representative two-photon images of the ear skin of mice systemically expressing GCaMP3 (GCaMP3 mice). (a) Three-dimensional view, (b) Vertical view showing cells with elevated fluorescence of GCaMP3 (GCaMP3^high cells). Arrow and arrowhead: GCaMP3^high cells in the basal and granular layers, respectively. Green: GCaMP3; red: autofluorescence; blue: second-harmonic generation from dermal collagen. Dashed line in (b): dermo-epidermal junction. (c) Hematoxylin and eosin staining of the ear skin section. Double-headed arrows: epithelial layers. (d) Horizontal view of the basal layer. Arrows: GCaMP3^high basal cells. (e) Maximum intensity view of the granular layer. Arrowhead: a GCaMP3^high granular cell. Arrow: nucleus. Scale bars: 50 μm (a–d) and 10 μm (e). [Ca²⁺]_i: concentration of calcium ions in the cytoplasm. Results are representative of at least three independent experiments.