Nonradioactive In Situ Hybridization of RNA Probes to Sections of Plant Tissues
This protocol was adapted from “How to Study Gene Expression,” Chapter 7, in Arabidopsis (eds. Weigel and Glazebrook). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.INTRODUCTION
Both radioactive and nonradioactive probes can be used to detect RNA in situ. Radioactive in situ hybridizations are believed by many to be more sensitive, but the localization of signal is considerably less precise than for nonradioactive in situs, and each experiment takes much longer. Thus, nonradioactive in situ hybridization is generally preferred. Antisense RNA is typically labeled using digoxygenin (DIG), which is subsequently detected with an anti-DIG antibody coupled to alkaline phosphatase. This protocol describes in situ hybridization of a DIG-labeled probe to paraffin-embedded sections of plant tissue. Double labeling using both DIG- and fluorescein isothiocyanate (FITC)-conjugated ribonucleotides is also described.
