In situ hybridization to mRNA of Arabidopsis tissue sections

Abstract

In situ hybridization detection of mRNA is an essential tool for understanding regulation of gene expression in cells and tissues of different organisms. Over the years, numerous in situ protocols have been developed ranging from whole-mount techniques that allow fast transcript localization in intact organs to high-resolution methods based on the electron microscopic detection of mRNAs at the subcellular level. Here, we present a detailed protocol for the detection of mRNAs in plant tissues using radiolabeled single-stranded RNA probes. Hybridizations are carried out on tissue sections of paraffin- and plastic-embedded plant tissues. Although this in situ protocol is appropriate for plant tissues in general, it has been optimized for Arabidopsis thaliana. Variations on the procedure, required to obtain optimal results with different Arabidopsis tissues, are described.