Protocol

Whole-Mount In Situ Hybridization of RNA Probes to 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

The labeling of entire plantlets (whole-mount labeling) is very easy, but it can cause substantial artifacts. Because whole plants are labeled, certain tissues are not readily accessible to the probes, whereas others can often stain nonspecifically. In the hands of the author, for instance, the elongation zone of the root is labeled even in negative controls, whereas mature parts of the roots and hypocotyl tend to be more resistant to probe penetration. Whole-mount labeling should therefore only be used to monitor gene expression in a limited number of organs and tissues--in particular, root meristems, embryos, and very young primordia--and the results should be interpreted with care. A protocol for whole-mount labeling is described here. Positive (e.g., constitutive probe) and negative controls should be included systematically.