Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced Tyramide Signal Amplification and Its Combination with Immunofluorescent Protein Visualization in Zebrafish

  • Gilbert Lauter
  • Iris Söll
  • Giselbert HauptmannEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


Fluorescent in situ hybridization (FISH) provides sensitive detection and visualization of RNA transcripts in tissues and cells with high resolution. We present here a multiplex RNA FISH method using enhanced tyramide signal amplification (TSA) for colocalization analysis of three different transcripts in intact zebrafish brains. To achieve enhancement of fluorescent signals, essential steps of the FISH procedure are optimized including embryo permeability, hybridization efficacy, and fluorogenic TSA-reaction conditions. Critical to this protocol, the enzymatic peroxidase (PO) reactivity is significantly improved by the application of viscosity-increasing polymers, PO accelerators, and highly effective bench-made tyramide substrates. These advancements lead to an optimized TSA–FISH protocol with dramatically increased signal intensity and signal-to-background ratio allowing for visualization of three mRNA transcript patterns simultaneously. The TSA–FISH procedure can be combined with immunofluorescence (IF) to compare mRNA transcript and protein expression patterns.


Fluorescent in situ hybridization FISH Tyramide signal amplification TSA Peroxidase Zebrafish Transcript RNA Immunofluorescence 



Imaging was in part performed at the Live Cell Imaging unit, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, and the Centre for Biosciences.


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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Gilbert Lauter
    • 1
  • Iris Söll
    • 2
  • Giselbert Hauptmann
    • 2
    Email author
  1. 1.Department of Biosciences and Nutrition, NeoKarolinska InstitutetHuddingeSweden
  2. 2.Department of Molecular Biosciences, The Wenner-Gren Institute, MBWStockholm UniversityStockholmSweden

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