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. 2019 Oct 10;9:14656. doi: 10.1038/s41598-019-51084-1

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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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Triple immunofluorescence labeling of Spleen (panels 1) and Granuloma sections (panels 2) from 127 S-infected animals. Nine-micrometer-thick slices were cut on a cryostat, fixed in 5% PFA, permeabilized in 0.5% X-100 Triton, blocked with 5% Bovine serum albumin and 2% rat and mouse sera, followed by the avidin-biotin blocking reaction. Incubation was performed with antibodies against CD45 (rabbit anti CD45 serum, gray, panels A) PrP^C (biotinylated Sha31, green, panels B), and mfge-8 (FDC-M1 rat monoclonal antibody, red, panels C). Incubation was followed with, anti-rat-Cy3 + streptavidin-Alexa-488 + anti-rabbit-Alexa 645 labeling, slide mounting using fluoromount and acquisition under a mono CCD camera. Panels D1 and D2 show the merged image built with the 4 channels (images in false colors: blue, DAPI counterstaining). Panels E1 and E2 correspond to spleen and granuloma sections stained using IgG_2c isotype control and secondary antibodies as staining control. Bars: 10 µm. Yellow arrows show PrP⁺/mfge-8⁺ cells that do not stain positive for CD45.