Abstract
The zebrafish Danio rerio is a valuable and common model for scientists in the fields of genetics and developmental biology. Since zebrafish are also amenable to genetic manipulation, modelling of human diseases or behavioral experiments have moved into the focus of zebrafish research. Consequently, gene expression data beyond embryonic and larval stages become more important, yet there is a dramatic knowledge gap of gene expression beyond day four of development. Like in other model organisms, the visualization of spatial and temporal gene expression by whole mount in situ hybridization (ISH) becomes increasingly difficult when zebrafish embryos develop further and hence the growing tissues become dense and less permeable. Here we introduce a modified method for whole mount ISH, which overcomes these penetration and detection problem. The method is an all in one solution that enables the detection and visualization of gene expression patterns up to the late larval stage in a 3D manner without the need for tissue sectioning and offers a valuable extension for whole mount ISH by immunohistochemistry in the zebrafish field.
Highlights
Whole mount in situ hybridization (WISH) detection of expressed mRNAs is an invaluable tool for research and diagnostics and a powerful application to study the cell- and tissue-specific expression of many genes in an organism
The main advantage of whole mount in situ hybridization (WISH) is that it is a quick and reliable method to determine the spatial distribution of specific mRNAs and the temporal gene expression in tissues
The optimal size range for whole mount ISH lies between embryonic day 6–9 [18]
Summary
Whole mount in situ hybridization (WISH) detection of expressed mRNAs is an invaluable tool for research and diagnostics and a powerful application to study the cell- and tissue-specific expression of many genes in an organism. The visualization of the spatial distribution of gene transcripts in single cells, tissues and embryos provides important information regarding developmental processes, health and disease [4,5,6]. The zebrafish Danio rerio has emerged as a useful model system for vertebrates because of a susceptibility to many tractable techniques for analysis of gene expression and function in whole mounts. Based on the excellent WISH protocol for zebrafish [7], many patterns of expressed genes have been published in high-resolution in embryos and early larvae [8,9].
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