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J Histochem Cytochem. 2019 Jan 9:22155418824335. doi: 10.1369/0022155418824335. [Epub ahead of print]

Gold-Substituted Silver-Intensified Peroxidase Immunolabeling for FIB-SEM Imaging.

Author information

1
Institute of Molecular and Cell Biology-Institute of Medical Biology Joint Electron Microscopy Suite, Agency for Science, Technology and Research, Singapore, Singapore.
2
Laboratory of Immunobiology, Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium.
3
VIB-KU Leuven Center for Brain & Disease Research, Electron Microscopy Platform & VIB-Bioimaging Core, Leuven, Belgium.
4
Department of Neurosciences, Leuven Brain Institute, KU Leuven, Leuven, Belgium.

Abstract

Modern electron microscopy offers a wide variety of tools to investigate the ultrastructural organization of cells and tissues and to accurately pinpoint intracellular localizations of macromolecules of interest. New volumetric electron microscopy techniques and new instrumentation provide unique opportunities for high-throughput analysis of comparatively large volumes of tissue and their complete reconstitution in three-dimensional (3D) electron microscopy. However, due to a variety of technical issues such as the limited penetration of label into the tissue, low antigen preservation, substantial electron density of secondary detection reagents, and many others, the adaptation of immuno-detection techniques for use with such 3D imaging methods as focused ion beam-scanning electron microscopy (FIB-SEM) has been challenging. Here, we describe a sample preparation method for 3D FIB-SEM, which results in an optimal preservation and staining of ultrastructural details at a resolution necessary for tracing immunolabeled neuronal structures and detailed reconstruction of synapses. This technique is applicable to neuronal and nonneuronal cells, tissues, and a wide variety of antigens.

KEYWORDS:

DAB; FIB-SEM; GSSP; brain; labeling

PMID:
30624131
DOI:
10.1369/0022155418824335

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