Abstract
Examination of spatial and temporal gene expression pattern is a key step towards understanding gene function. Therefore, in situ hybridization of mRNA is one of the most powerful and widely used techniques in biology. Recent advances allow the reliable and simultaneous detection of mRNA transcripts, or combinations of mRNA and protein, in zebrafish embryos.
Here we describe a standard protocol for visualizing the precise expression pattern of a single transcript or multiple gene products. The procedure employs fixation and permeabilization of embryos, followed by hybridization with tagged antisense riboprobes. Excess probes are then washed and hybrids are detected by enzyme-mediated immunohistochemistry utilizing either chromogenic or fluorescent substrates.
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Acknowledgments
We are grateful to Nataliya Borodovsky and Amos Gutnick for stimulating discussion and comments on this manuscript. Nataliya Borodovsky also provided the ISH images. The research in the Levkowitz lab is supported by the German-Israeli Foundation (grant number 183/2007); Israel Science Foundation (grant number 928/08) and the Harriet & Marcel Dekker Foundation. G.L. is an incumbent of the Tauro Career Development Chair in Biomedical Research.
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Machluf, Y., Levkowitz, G. (2011). Visualization of mRNA Expression in the Zebrafish Embryo. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_6
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DOI: https://doi.org/10.1007/978-1-61779-005-8_6
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