In situ Hybridization

Abstract

In situ hybridization techniques enable gene expression to be monitored in individual cells (Figure 18.1). Methods for use with the light microscope are as sensitive as northern blot analysis for detecting mRNAs that accumulate equally in all cells, and more sensitive than northern analysis for detecting mRNAs that are present in only a small population of cells within an organ (Langdale et al. 1988a,b; Martineau and Taylor 1986). The following protocol has been used most frequently to monitor gene expression in maize leaf tissue. By varying tissue fixation conditions, all organs of the maize plant have been analyzed. The method uses paraffin-embedded sections and ³⁵S-radiolabeled or digoxigenin nonradiolabeled riboprobes. The use of nonradiolabeled probes is a fairly recent advance and at this stage they appear to be less sensitive than radiolabeled probes. However, the method can undoubtedly be improved with further use and optimization. Tissue that is fixed and embedded as described below may also be used for immunocytochemistry. Standard methods of microscopy and photomicroscopy are used for signal detection. See appropriate chapters in this volume for details.