Whole-mount in situ hybridization is the preferred method for detecting transcript distributions in whole embryos, tissues, and organs. We present here a sensitive fluorescent in situ hybridization method for colocalization analysis of different transcripts in whole embryonic zebrafish brains. The method is based on simultaneous hybridization of differently hapten-labeled RNA probes followed by sequential rounds of horseradish peroxidase (POD)-based transcript detection. Sequential detection involves enhancement of fluorescent signals by tyramide signal amplification (TSA) and effective inactivation of the antibody–POD conjugate prior to the following detection round. We provide a detailed description of embryo preparation, hybridization, antibody detection, POD–TSA reaction, and mounting of embryos for imaging. To achieve high signal intensities, we optimized key steps of the method. This includes improvement of embryo permeability by hydrogen peroxide treatment and efficacy of hybridization and TSA–POD reaction by addition of the viscosity-increasing polymer dextran sulfate. The TSA–POD reaction conditions are further optimized by application of substituted phenol compounds as POD accelerators and use of highly efficient bench-made tyramide substrates. The obtained high signal intensities and cellular resolution of our method allows for co-expression analysis and generation of three-dimensional models. Our protocol is tailored to optimally work in zebrafish embryos, but can surely be modified for application in other species as well.