Multicolor 3D Fluorescence In Situ Hybridization for Imaging Interphase Chromosomes

Fluorescence in situ hybridization (FISH) of specifi c DNA probes has become a widely used technique mostly for chromosome analysis and for studies of the chromosomal location of specifi c DNA segments in metaphase prepara- tions as well as in interphase nuclei. FISH on 3D-preserved nuclei (3D-FISH) in combination with 3D-microscopy and image reconstruction is an effi cient tool to analyze the spatial arrangement of targeted DNA sequences in the nucleus. Recent developments of a "new generation" of confocal microscopes that allow the distinct visualization of at least fi ve different fl uorochromes within one experiment opened the way for multicolor 3D-FISH experiments. Thus, numerous differently labeled nuclear targets can be delineated simultaneously and their spatial interrelationships can be analyzed on the level of individual nuclei. In this chapter, we provide protocols for the preparation of complex DNA-probe sets suitable for 3D-FISH with up to six different fluorochromes, for 3D-FISH on cultured mammalian cells (growing in suspension or adherently) as well as on tis- sue sections, and for 3D immuno-FISH. In comparison with FISH on metaphase chromosomes and conventional inter- phase cytogenetics, FISH on 3D-preserved nuclei requires special demands with regard to probe quality, fixation, and pretreatment steps of cells in order to achieve the two goals, namely the best possible preservation of the nuclear structure and at the same time an efficient probe accessibility.