High Resolution Whole Mount <em>In Situ</em> Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Babykumari P. Chitramuthu; Hugh P. J. Bennett

doi:10.3791/50644

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene...

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High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

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10:06 min

October 19, 2013

DOI: 10.3791/50644-v

Babykumari P. Chitramuthu^1,2, Hugh P. J. Bennett^1,2

¹Endocrine Research Laboratory and Department of Medicine,Royal Victoria Hospital, ²McGill University Health Centre Research Institute

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

The overall goal of this procedure is to assess the spatial temporal expression of specific genes of interest within staged zebrafish embryos. This is accomplished by first generating CD NA probes by incorporating digo oxygen in linked nucleotides using in vitro transcription. Next, the embryos are harvested at defined developmental stages.

The corium are removed and the embryos are stored in methanol at negative 20 degrees Celsius. Then following digestion and post fixation, the embryos are hybridized with the appropriate probes. Finally, the embryos are stained with antide gene and antibodies and the signal developed with substrates for alkaline phosphatase.

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