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Gold-substituted Silver-intensified Peroxidase Immunolabeling for FIB-SEM Imaging.

Authors :
Boey A
Rybakin V
Kalicharan D
Vints K
Gounko NV
Source :
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society [J Histochem Cytochem] 2019 May; Vol. 67 (5), pp. 351-360. Date of Electronic Publication: 2019 Jan 09.
Publication Year :
2019

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 non-neuronal cells, tissues, and a wide variety of antigens.

Details

Language :
English
ISSN :
1551-5044
Volume :
67
Issue :
5
Database :
MEDLINE
Journal :
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society
Publication Type :
Academic Journal
Accession number :
30624131
Full Text :
https://doi.org/10.1369/0022155418824335