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. 2019 Mar 29;9:5361. doi: 10.1038/s41598-019-41779-w

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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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Illustration of the method used for the analysis of nerve profiles in human bone. (a) Masson’s trichrome stained section showing the overall compartmentalization of the iliac crest biopsy into bone marrow, cortical bone, and periosteum. Sections were sequentially immunostained for tyrosine hydroxylase (TH, black arrows) and protein gene product 9.5 (PGP9.5, red arrows). The images illustrate this immunostaining within the bone marrow (b), cortical pore (c) and periosteum (d). The sequential immunostaining allows discrimination between the TH+ nerve profiles with sympathetic nerve fibers (brown) and the TH−/PGP9.5+ nerve profiles without sympathetic nerve fibers (red). Scale bars are 200 µm (a) and 50 µm (b–d).