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

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

High-resolution in situ hybridization to whole-mount zebrafish embryos Christine Thisse Bernard Thisse Department of Cell Biology, University of Virginia Health Sciences Center, PO Box 800732, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22908, USA. Correspondence should be addressed to C.T. (christhisse@) or B.T. (bernardthisse@). Published online 20 December 2007; doi:10.1038/nprot.2007.514 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. INTRODUCTION The ISH method allows specific nucleic acid sequences to be detected in morphologically preserved cells or embryos. It relates microscopic topological information to gene activity at the DNA or mRNA level. ISH to whole-mount embryos is a method widely used to describe the expression pattern of developmentally regulated genes. Overview of ISH Original methods for ISH developed in the 1980s, using radio- actively labeled DNA probes to detect expression of genes on histological sections, have been widely used in many model organisms including zebrafish1. In the 1990s, the use of chemically lab