Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5019

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High-Resolution Fluorescent In Situ Hybridization of Drosophila Embryos and Tissues

Eric Lécuyer, Aleksandar S. Necakov, Lucía Cáceres, and Henry M. Krause1

Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 3E1, Canada

1Corresponding author (h.krause{at}utoronto.ca)


INTRODUCTION

Fluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs in intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases the sensitivity and resolution of FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, and larval tissues. Instructions are given for the preparation of RNA probes, the collection and fixation of tissues, and the hybridization and TSA-mediated detection of probes, including options for high-throughput processing in 96-well plates. Variations of the procedure for RNA-RNA and RNA-protein costaining are also described.


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