Methodology

3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei

Leïla Tirichine^1,6 , Philippe Andrey^2,3,4,5 , Eric Biot¹ , Yves Maurin^2,3,4 and Valérie Gaudin¹

¹  Laboratoire de Biologie Cellulaire, INRA UR 501, IJPB, Route de Saint-Cyr, F-78026 Versailles, France

²  Neurobiologie de l'Olfaction et de la Prise Alimentaire, INRA UMR 1197, Domaine de Vilvert, F-78350 Jouy-en-Josas, France

³  Université Paris-Sud 11, UMR 1197, F-91400 Orsay, France

⁴  IFR 144 Neuro-Sud, Paris, France

⁵  Université Pierre et Marie Curie, Paris, France

⁶  Institut des Sciences du Végétal, CNRS, avenue de la Terrasse, F-91198 Gif-sur-Yvette, France

author email corresponding author email

Plant Methods 2009, 5:11doi:10.1186/1746-4811-5-11

Published:	3 August 2009

Abstract

Background

Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill.

Results

We report a rapid protocol for fluorescent in situ hybridization (FISH) performed on 3D isolated nuclei and thin cryosectioned leaves of Arabidopsis thaliana. The use of direct labeling minimized treatment steps, shortening the overall procedure. Using image analysis, we measured different parameters related to nucleus morphology and overall 3D structure.

Conclusion

Our work describes a 3D-FISH protocol that preserves the 3D structure of Arabidopsis interphase nuclei. Moreover, we report for the first time FISH using cryosections of Arabidopsis leaves. This protocol is a valuable tool to investigate nuclear architecture and chromatin organization.