Immunolabel-First-Expand-Later Expansion Microscopy Approach Using Stable STED Dyes.
Abberior STAR ORANGE
Abberior STAR RED
Centriole
Centrosome
Cep164
Cilia
Expansion
STED
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2024
2024
Historique:
medline:
1
11
2023
pubmed:
19
10
2023
entrez:
19
10
2023
Statut:
ppublish
Résumé
Multiple expansion microscopy approaches have been successfully used in the analysis of centrioles, centrosomes, and cilia, helping to reveal the localization of numerous centrosomal and ciliary proteins at nanoscale resolution. In this chapter, we describe the use of two stable STED dyes in combination with expansion microscopy, which allows the robust detection by conventional and STED microscopy of proteins immunolabeled prior to sample expansion. We demonstrate the stability of these dyes during the crosslinking, polymerization, and denaturation steps of an expansion protocol thereby allowing their use in an immunolabel-first-expand-later approach. Our protocol overcomes the frequent technical limitation of poor, unreproducible binding of primary antibodies to proteins after denaturation. We demonstrate the applicability of this approach by analyzing both a centriole appendage protein Cep164 and a ciliary protein ARL13B.
Identifiants
pubmed: 37856019
doi: 10.1007/978-1-0716-3507-0_5
doi:
Substances chimiques
Coloring Agents
0
Antibodies
0
Types de publication
Journal Article
Research Support, N.I.H., Intramural
Langues
eng
Sous-ensembles de citation
IM
Pagination
89-101Informations de copyright
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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