An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies.
RNA fluorescence in situ hybridization
glyoxal
immunofluorescence
nuclear body
paraformaldehyde
Journal
RNA (New York, N.Y.)
ISSN: 1469-9001
Titre abrégé: RNA
Pays: United States
ID NLM: 9509184
Informations de publication
Date de publication:
06 2021
06 2021
Historique:
received:
29
12
2020
accepted:
08
04
2021
pubmed:
14
4
2021
medline:
24
9
2021
entrez:
13
4
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33846273
pii: rna.078671.120
doi: 10.1261/rna.078671.120
pmc: PMC8127994
doi:
Substances chimiques
Fixatives
0
Polymers
0
Formaldehyde
1HG84L3525
Glyoxal
50NP6JJ975
paraform
Y19UC83H8E
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
725-733Subventions
Organisme : Howard Hughes Medical Institute
ID : 55008728
Pays : United States
Informations de copyright
© 2021 Yao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
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