Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae
- He, J., Mo, D., Chen, J., Luo, L.
- Nature Protocols 15: 3361-3379 (Other)
- Registered Authors
- He, Jianbo, Luo, Lingfei
- MeSH Terms
- Embryo, Nonmammalian/diagnostic imaging*
- Fluorescent Antibody Technique/methods*
- In Situ Hybridization, Fluorescence/methods*
- RNA, Messenger/genetics
- 32908315 Full text @ Nat. Protoc.
He, J., Mo, D., Chen, J., Luo, L. (2020) Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae. Nature Protocols. 15:3361-3379.
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.
Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes