Osmium-Resistant Fluorescent Proteins and In-Resin Correlative Light-Electron Microscopy of Epon-Embedded Mammalian Cultured Cells.
Methods Mol Biol
; 2564: 287-297, 2023.
Article
em En
| MEDLINE
| ID: mdl-36107349
ABSTRACT
Postfixation with osmium tetroxide and Epon embedding are essential for the preservation and visualization of subcellular ultrastructures via electron microscopy. These chemical treatments diminish the fluorescent intensity of most fluorescent proteins in cells, creating a problem for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded mammalian cultured cells. We found that two green and two far-red fluorescent proteins retain their fluorescence after chemical fixation with glutaraldehyde, osmium tetroxide-staining, dehydration, and polymerization of Epon resins. Consequently, we could observe the fluorescence of fluorescent proteins in ultrathin sections of Epon-embedded cells via fluorescence microscopy, investigate ultrastructures of the cells in the same sections via electron microscopy, and correlate the fluorescent image with the electron microscopic image without chemical or physical distortion of the cells. In other words, referred as "in-resin CLEM" of Epon-embedded samples. This technique also improves the Z-axis resolution of fluorescent images. In this chapter, we introduce the detailed protocol for in-resin CLEM of Epon-embedded mammalian cultured cells using these fluorescent proteins.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Osmio
/
Tetróxido de Ósmio
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Japão
País de publicação:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
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US
/
USA