Examination of spatial and temporal gene expression pattern is a key step towards understanding gene function. Therefore,in situhybridization of mRNA is one of the most powerful and widely used �techniques in biology. Recent advances allow the reliable and simultaneous detection of mRNA transcripts, or combinations of mRNA and protein, in zebrafish embryos. Here we describe a standard protocol for visualizing the precise expression pattern of a single transcript or multiple gene products. The procedure employs fixation and permeabilization of embryos, �followed by hybridization with tagged antisense riboprobes. Excess probes are then washed and hybrids are detected by enzyme-mediated immunohistochemistry utilizing either chromogenic or fluorescent substrates.
