title: Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization.
abstract: In situ mRNA hybridization is one of the most powerful techniques for analyzing patterns of gene expression. However, its usefulness is limited in complex plant tissues by the need to fix, embed and section samples before localizing the desired mRNA. Here we present a robust whole-mount in situ hybridization method that allows easy access to patterns of gene expression in intact, complex tissues, such as the inflorescence apex of Arabidopsis thaliana. The tissue is first fixed and then permeabilized by treatment with a cocktail of cell wall-digesting enzymes that has been optimized to preserve the integrity of tissue structures, while also permitting the detection of expression patterns in deep tissues. In addition to colorimetric staining, fluorimetric staining that can be imaged by confocal microscopy can also be used with this protocol, thus providing full 3D resolution. The entire procedure can take <3 d from tissue preparation to image acquisition.
authors.affiliation: Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France.
authors.forename: Frédérique
authors.initials: F
authors.lastname: Rozier
authors.affiliation: 1] Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France. [2] Laboratoire Joliot Curie, Ecole Normale Supérieure Lyon, Lyon, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Mirabet
authors.affiliation: Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France.
authors.forename: Teva
authors.initials: T
authors.lastname: Vernoux
authors.affiliation: 1] Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France. [2] Laboratoire Joliot Curie, Ecole Normale Supérieure Lyon, Lyon, France.
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D003124:Colorimetry / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D018749:RNA, Plant
chemical_list: D018749:RNA, Plant