In situ hybridization to evaluate the expression of Wnt and frizzled genes in mammalian tissues.

In situ hybridization can be utilized to specifically define the expression of genes and to determine their localization in complex mammalian tissues. The expression of specific members of the Wnt ligand and frizzled receptor families of molecules can be defined using an antisense RNA probe that will specifically hybridize with messenger RNA (mRNA) in the tissue to form a double-stranded product. The double-stranded product can then be detected microscopically by identifying digoxigenin groups that are attached to the probe during its synthesis. Probe sequence selection is critical to ensure specificity among different Wnt or frizzled family members. Controls are needed at every step in the technique to confirm appropriate quality of the tissue sections, quality of the prepared probe, and specificity of the hybridization reactions. If performed properly, in situ hybridization can be utilized to define gene expression and specific localization of RNA in human and other mammalian tissues, and can be utilized in previously fixed and paraffin-embedded tissue samples.