Abstract
BackgroundWhole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.ResultsThe protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.ConclusionsBy fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.
Highlights
Whole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues
The signal generated in fluorescent in situ hybridization (FISH) relies on the binding of a horse radish peroxidase (HRP)-conjugated antibody to modified ribonucleotides of the probe, followed by tyramide signal amplification (TSA), where HRP converts a tyramide conjugated to a fluorophore into a reactive fluorescent intermediate that covalently binds to nearby amino-acid residues, typically tyrosine
Whereas multicolor FISH can potentially be used for detecting multiple transcripts by employing different fluorescent tyramide substrates, the method is less efficient, for RNAs expressed at low levels [6]
Summary
Whole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. Compared to chromogenic ISH, fluorescent in situ hybridization (FISH) offers a higher resolution and an improved detection of overlapping gene expression patterns in a single sample [5,6,7]. Whereas multicolor FISH can potentially be used for detecting multiple transcripts by employing different fluorescent tyramide substrates, the method is less efficient, for RNAs expressed at low levels [6]. To overcome this drawback of the method, modifications to the original FISH protocol have been put forward that significantly enhance the sensitivity of the assay [7,8].
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