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. 2018 Nov 9;8:16594. doi: 10.1038/s41598-018-34808-7

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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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Ubiquitinated and glycosylated p62/SQSTM1-immunopositive BD-inclusions exhibiting N-terminal epitopes of APP in cardiomyocytes in cross sections through the left ventricle wall. (A) BD-inclusion (arrows) stained with the PAS histochemical staining method in a cardiomyocyte of the left ventricular myocardium in case No. 53. (B) Subsequent section of the BD-inclusion of case No. 53 depicted in A immunostained with anti-APP (22C11) antibodies against the N-terminus of APP (arrows). Note that neighboring cardiomyocytes exhibit a physiological APP expression in the cytoplasm, focally associated with lipofuscin granules. (C) p62/SQSTM1- immunohistochemistry showed in another subsequent section of the BD-inclusion of case No. 53 depicted in (A,B) the presence of p62/SQSTM1 (arrows). The aggregates exhibited an amorphous structure and were sharply delineated. (D) The section shown in C was also stained for ubiquilin by double label immunofluorescence, which was colocalizing with p62/SQSTM1 in the BD-lesion (arrows). (E–H) Triple label immunofluorescence of a BD-lesion in the myocardium of the left ventricle of case No. 17. The BD-lesion was stained with anti-APP (22C11) detecting the N-terminus of APP (arrow in E,H) and p62/SQSTM1 (arrow in F,H) whereas the antibody against the D-epitope of the Aη-region did not detect this lesion (arrow in G,H). (I–L) Triple label immunofluorescence of a BD-lesion in case No. 50 shows expression of ubiquitin (arrow in I,L) and p62/SQSTM1 (arrow in J,L) but no expression of Aβ detected with the antibody (3552)⁴⁴ (arrow in K,L). (M–O) Double label immunofluorescence of a BD-lesion in the left ventricular myocardium of case No. 56 showed a p62/SQSTM1-positive BD inclusion (arrowhead in M,O) which was not visible with the antibody against APLP2 (arrowhead in N,O) whereas the cardiomyocytes showed a mild physiological cytoplasmic staining of APLP2. (P–R) Negative controls (myocardium of the left ventricle from case No. 17) for immunofluorescence stainings were performed by omitting primary antibodies (only secondary antibodies/detection agents): Carbocyanin-2 (Cy2)-conjugated streptavidin (P) Cy5-conjugated anti-mouse IgG (Q) and Cy3-conjugated anti-rabbit IgG secondary antibodies (R). Positive controls are depicted in Suppl. Fig. 1.