A rapid and sensitive, multiplex, whole mount RNA fluorescence in situ hybridization and immunohistochemistry protocol.
Fluorescent protein
Hybridization chain reaction
Immunohistochemistry
RNA-FISH
Whole mount
Journal
Plant methods
ISSN: 1746-4811
Titre abrégé: Plant Methods
Pays: England
ID NLM: 101245798
Informations de publication
Date de publication:
22 Nov 2023
22 Nov 2023
Historique:
received:
29
05
2023
accepted:
10
11
2023
medline:
23
11
2023
pubmed:
23
11
2023
entrez:
23
11
2023
Statut:
epublish
Résumé
In the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported. We adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol. The whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.
Sections du résumé
BACKGROUND
BACKGROUND
In the past few years, there has been an explosion in single-cell transcriptomics datasets, yet in vivo confirmation of these datasets is hampered in plants due to lack of robust validation methods. Likewise, modeling of plant development is hampered by paucity of spatial gene expression data. RNA fluorescence in situ hybridization (FISH) enables investigation of gene expression in the context of tissue type. Despite development of FISH methods for plants, easy and reliable whole mount FISH protocols have not yet been reported.
RESULTS
RESULTS
We adapt a 3-day whole mount RNA-FISH method for plant species based on a combination of prior protocols that employs hybridization chain reaction (HCR), which amplifies the probe signal in an antibody-free manner. Our whole mount HCR RNA-FISH method shows expected spatial signals with low background for gene transcripts with known spatial expression patterns in Arabidopsis inflorescences and monocot roots. It allows simultaneous detection of three transcripts in 3D. We also show that HCR RNA-FISH can be combined with endogenous fluorescent protein detection and with our improved immunohistochemistry (IHC) protocol.
CONCLUSIONS
CONCLUSIONS
The whole mount HCR RNA-FISH and IHC methods allow easy investigation of 3D spatial gene expression patterns in entire plant tissues.
Identifiants
pubmed: 37993896
doi: 10.1186/s13007-023-01108-9
pii: 10.1186/s13007-023-01108-9
pmc: PMC10666358
doi:
Types de publication
Journal Article
Langues
eng
Pagination
131Subventions
Organisme : Human Frontiers of Science
ID : LT000972/2018-L
Organisme : Division of Integrative Organismal Systems
ID : 2319036
Informations de copyright
© 2023. The Author(s).
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