Abstract
Biological material itself appears with poor contrast in electron microscopy (EM), due to its composition mostly of light elements. Classical staining agents such as osmium tetroxide, uranyl acetate, and lead citrate preserve and/or stain cellular structures such as membranes, cytoplasm, and organelles well for EM. However, extracellular polymeric substances (EPS) show no or only poor contrast with these staining agents. The endothelial glycocalyx in blood vessels consists mainly of proteoglycans. It can be visualized by EM only by additional staining with heavy metal ions such as copper (Alcian blue, cupromeronic blue), ruthenium (ruthenium red), or lanthanum. Best results are achieved by combined perfusion of fixative and stain. Cationic hydrous thorium dioxide colloids (named here cThO2) trace acidic groups in EPS. We describe here the use of cThO2 to visualize the glomerular endothelial glycocalyx in the mouse kidney. cThO2 shows high electron density and binds to a continuous layer of up to a few hundred nanometers thickness on the glomerular endothelium, as well as on epithelia in other blood vessels in perfused animals. The observed staining pattern gives rise to periodic densities, with a spacing varying between 50 and 200 nm, depending on the overall layer thickness, which varies between below 50 up to 300 nm. Due to high electron density of the used cThO2 particles, the introduced method allows distinct imaging and precise fine structural analysis of the endothelial glycocalyx.
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Acknowledgments
This study was supported by the German Research Federation (REBIRTH Cluster of Excellence). We thank Dr. Christoph Wrede (Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany) for excellent assistance in electron microscopy, and Herle Chlebusch, Hayet Richy (Department of Nephrology, Hannover Medical School, Hannover, Germany), and Inge Kristen (ZEIM, Helmholtz Center for Infection Research, Braunschweig, Germany) for excellent technical assistance.
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JH and HL performed the experiments. JH, MO, and HH designed the study. JH drafted the manuscript. All authors have read and approved the final manuscript.
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All applicable international, national, and/or international guidelines for the care and use of animals were followed.
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Movie S1
The movie shows the tomographic 3D model shown in Figure 4 from different viewing angles. Biological material is displayed in green and thorium dioxide in red. The lumen of the glomerular capillary faces upwards and the podocytes downwards. The GBM appears translucent. (MP4 1124 kb)
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Hegermann, J., Lünsdorf, H., Ochs, M. et al. Visualization of the glomerular endothelial glycocalyx by electron microscopy using cationic colloidal thorium dioxide. Histochem Cell Biol 145, 41–51 (2016). https://doi.org/10.1007/s00418-015-1378-3
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DOI: https://doi.org/10.1007/s00418-015-1378-3