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. 2018 Mar 16;8:4711. doi: 10.1038/s41598-018-22934-1

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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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Expression of PDE4 (NPP4) protein within nerve fibers distributed among human bladder neck smooth muscle bundles. Double-labeling immunofluorescence assay in the human bladder neck (A–D). Bladder neck overall innervation was visualized using the general nerve marker PGP 9.5 (green areas) (A). NPP4 immunofluorescence from human bladder neck reveals immunopositive nerve trunks (red areas), running parallel to the smooth muscle bundles. Same fields (A,B,E and F). Immunofluorescence double-labeling for PGP 9.5 and NPP4 in the smooth muscle, demonstrate neuronal co-localization (yellow areas) (C). Cell nuclei were counterstained with DAPI (blue areas) (D). Scale bars indicate 25 µm. Negative controls showing the lack of immunoreactivity in sections incubated in the absence of the primary antibody (E and F) (n = 4). Comparison of the fluorescence of PGP 9.5 and NPP4, using ImageJ, which shows a priority co-localization between NPP4 and PGP 9.5 (G). Western blot of smooth muscle membranes from human bladder neck incubated with NPP4 antibody showing a 52 kDa major band, which corresponded to the expected molecular weight (H) (n = 4).