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. 2017 Aug 17;7:8580. doi: 10.1038/s41598-017-08719-y

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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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GFP⁺ cells in muscle or respiratory tract after IM injection or IN instillation of rMVA-GFP to mice. (a) Maximum intensity projection of a Z-stack of the hind leg muscle 24 h after IM injection of rMVA-GFP. Green = GFP. Red = nucleus (stained by TO-PRO3). (b) Detection of GFP⁺ myocytes in hind leg muscle tissue after IM rMVA-GFP injection by staining with rabbit anti-GFP or rabbit Ig isotype (green) in combination with DAPI (blue). (c) Morphological characterization of GFP⁺ cells in hind leg muscle tissue after IM rMVA-GFP injection by CLSM (left two panels) or IHC (right two panels) showing hind leg muscle (24 HPA) or negative control front leg muscle (6 HPA). GFP⁺ interdigitating cells are indicated by arrows. CLSM: GFP = green, nucleus = blue (DAPI). IHC: GFP = red and counterstaining with haematoxylin. (d) Detection of GFP⁺ cells in the nasal turbinates and lungs 6 or 24 h after IN instillation of rMVA-GFP. GFP⁺ cells are shown in red (indicated by arrows). Tissues were counterstained with haematoxylin. (e) Morphological characterization of GFP⁺ cells in lungs after IN instillation of rMVA-GFP by staining with rabbit anti-GFP or rabbit Ig isotype (green) in combination with DAPI (blue). GFP⁺ cell with macrophage-/DC-like morphology is indicated by arrow. (f) Percentage of GFP⁺ cells within different cell populations 6 h after IN instillation of 10⁷, 10⁸ or 10⁹ PFU rMVA-GFP determined by flow cytometry. Results are shown as mean of two mice. (g) Relative contribution of the different cellular subsets to the GFP⁺ population by reversed gating. The average percentages of all mice that received rMVA-GFP by IN instillation and were euthanized 6 HPA are shown. IMφ = interstitial macrophages. Contrast of some CLSM images have been linearly enhanced using Adobe Photoshop CC. Scale bars are indicated in each figure.