Visualizing the translational activation of a particular mRNA in zebrafish embryos using in situ hybridization and proximity ligation assay

Fertilized eggs initiate translation of stored mRNAs in spatially and temporally controlled manners. Here, we present a protocol for visualizing spatial and temporal translation in zebrafish embryos by fluorescence in situ hybridization and proximity ligation assay. We describe steps for labeling newly synthesized proteins and mRNA, visualizing mRNA translation and mRNA, sample mounting, and observation. Coupling detection of mRNA molecules with their translation sites is useful for understanding the molecular and cellular mechanisms that drive embryo development. For complete details on the use and execution of this protocol, please refer to Sato et al.1 and Takada et al.2 [Display omitted] •Protocol for visualizing translational activation of mRNAs in zebrafish embryo•Optimized approach for ribopuromycylation and proximity ligation assay•Detecting the translation sites by in situ hybridization and proximity ligation assay•Imaging by confocal and super-resolution microscopes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Fertilized eggs initiate translation of stored mRNAs in spatially and temporally controlled manners. Here, we present a protocol for visualizing spatial and temporal translation in zebrafish embryos by fluorescence in situ hybridization and proximity ligation assay. We describe steps for labeling newly synthesized proteins and mRNA, visualizing mRNA translation and mRNA, sample mounting, and observation. Coupling detection of mRNA molecules with their translation sites is useful for understanding the molecular and cellular mechanisms that drive embryo development.