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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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Identification of rMVA-GFP⁺ cells in macaque muscle or lung after IM injection or AER inhalation, respectively. (a) Detection of GFP⁺ cells at site of rMVA-GFP administration. Left panel: hind leg muscle after IM injection stained with rabbit anti-GFP (green, indicated by arrows) and DAPI (blue). Right panel: direct detection of GFP fluorescence in lung slices after AER inhalation counterstained with TO-PRO3 (red). Maximum intensity projection of a Z-stack is shown. (b) Dual immunofluorescent staining of muscle slices after IM injection of rMVA-GFP with rabbit anti-GFP (green) in combination with DAPI (blue) and mouse anti-Mac387, anti-MHC class II or anti-desmin (red). Co-localization of GFP and desmin is indicated by an arrow. (c) Dual immunofluorescent staining of lung slices after AER inhalation of rMVA-GFP with rabbit anti-GFP (green) in combination with DAPI (blue) and mouse anti-cytokeratin, anti-CD20, anti-Mac387, anti-MHC class II or anti-CD11c (red). Co-localization of GFP and MHC class II or CD11c is indicated by arrows. (d) Lung slices stained with isotype control antibodies (rabbit Ig as control for anti-GFP, mouse IgG1 and 2a as control for cytokeratin, CD20, Mac387, MHC class II, CD11c, CD3 and desmin). (e) Percentage of GFP⁺ BAL cells within different cell populations after IM injection or AER inhalation of rMVA-GFP determined by flow cytometry. Mean of two macaques is indicated. (f) Relative contribution of the different subsets to the GFP⁺ population determined by reversed gating. The averages of the two animals that received rMVA-GFP by AER are shown. Contrast of some CLSM images has been enhanced linearly using Adobe Photoshop CC. Scale bars are indicated in images.