Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae.
Journal
Nature protocols
ISSN: 1750-2799
Titre abrégé: Nat Protoc
Pays: England
ID NLM: 101284307
Informations de publication
Date de publication:
10 2020
10 2020
Historique:
received:
25
04
2019
accepted:
19
06
2020
pubmed:
11
9
2020
medline:
4
11
2020
entrez:
10
9
2020
Statut:
ppublish
Résumé
RNA fluorescence in situ hybridization (FISH) and antibody staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs and proteins. Here we describe a combined FISH and IF protocol to simultaneously detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. In our approach, FISH is performed before IF to prevent mRNA degradation during the IF procedure. Instead of proteinase K digestion, Triton X-100 treatment and skin removal are used to permeate tissues and preserve antigen epitopes, making this protocol applicable to both whole-mount embryos and larvae. Off-target hybridization and FISH background are reduced by using PCR-amplified DNA templates and stringent buffers. This protocol simultaneously detects multiple mRNAs and proteins with high sensitivity, and enables detection at single-cell resolution. The protocol can be completed within 6 days, overcoming the shortage of reliable antibodies available for zebrafish and exploiting the advantages of zebrafish for studying organ development and regeneration.
Identifiants
pubmed: 32908315
doi: 10.1038/s41596-020-0376-7
pii: 10.1038/s41596-020-0376-7
doi:
Substances chimiques
Antibodies
0
RNA, Messenger
0
RNA
63231-63-0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
3361-3379Références
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