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High-resolution in situ hybridization to whole-mount zebrafish embryos


The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.

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Figure 1: Expression pattern of NcoR, a nuclear receptor corepressor.
Figure 2: Flow diagram.
Figure 3: Baskets and boxes used for rehydration, incubation and washes.
Figure 4: Mounting stained embryos for observation and imaging.
Figure 5: Rehydration, proteinase K digestion, post-fixation and washes.
Figure 6: Monitoring the staining reaction embryos in staining solution are placed in a spot plate and the reaction is monitored using a stereomicroscope, illuminated from above.
Figure 7: Anticipated results.


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This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, the European Commission as part of the ZF-Models integrated project in the 6th Framework Programme and the National Institute of Health (R01 RR15402).

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Correspondence to Christine Thisse or Bernard Thisse.

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Thisse, C., Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc 3, 59–69 (2008).

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