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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
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