An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies.
RNA
; 27(6): 725-733, 2021 06.
Article
em En
| MEDLINE
| ID: mdl-33846273
ABSTRACT
The mammalian cell nucleus contains different types of membrane-less nuclear bodies (NBs) consisting of proteins and RNAs. Microscopic imaging has been widely applied to study the organization and structure of NBs. However, current fixation methods are not optimized for such imaging When a fixation method is chosen to maximize the quality of the RNA fluorescence in situ hybridization (FISH), it often limits the labeling efficiency of proteins or affects the ultrastructure of NBs. Here, we report that addition of glyoxal (GO) into the classical paraformaldehyde (PFA) fixation step not only improves FISH signals for RNAs in NBs via augmented permeability of the fixed nucleus and enhanced accessibility of probes, but also largely preserves protein fluorescent signals during fixation and immunostaining. We also show that GO/PFA fixation enables the covisualization of different types of nuclear bodies with minimal impact on their ultrastructures under super-resolution microscopy.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polímeros
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Hibridização in Situ Fluorescente
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Estruturas do Núcleo Celular
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Fixadores
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Formaldeído
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Glioxal
Limite:
Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article