|ZFIN ID: ZDB-PUB-201002-7|
Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae
He, J., Mo, D., Chen, J., Luo, L.
|Source:||Nature Protocols 15: 3361-3379 (Other)|
|Registered Authors:||He, Jianbo, Luo, Lingfei|
|PubMed:||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.
ABSTRACTRNA 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.
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