Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography.
Traffic
; 19(8): 639-649, 2018 08.
Article
em En
| MEDLINE
| ID: mdl-29673018
ABSTRACT
Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.
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Base de dados:
MEDLINE
Assunto principal:
Tomografia
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Microscopia Imunoeletrônica
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Microscopia Eletrônica de Transmissão
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article