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.
Date: 2020
Source: Nature Protocols   15: 3361-3379 (Other)
Registered Authors: He, Jianbo, Luo, Lingfei
Keywords: none
MeSH Terms:
  • Animals
  • Antibodies/metabolism
  • Embryo, Nonmammalian/diagnostic imaging*
  • Fluorescent Antibody Technique/methods*
  • In Situ Hybridization, Fluorescence/methods*
  • Larva/metabolism
  • RNA/metabolism
  • RNA, Messenger/genetics
  • Zebrafish/genetics
PubMed: 32908315 Full text @ Nat. Protoc.
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.