Multicolor 3D Fluorescence In Situ Hybridization for Imaging Interphase Chromosomes

Abstract

Fluorescence in situ hybridization (FISH) of specific DNA probes has become a widely used technique mostly for chromosome analysis and for studies of the chromosomal location of specific DNA segments in metaphase preparations as well as in interphase nuclei. FISH on 3D-preserved nuclei (3D-FISH) in combination with 3D-microscopy and image reconstruction is an efficient 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 five different fluorochromes 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 tissue sections, and for 3D immuno-FISH.In comparison with FISH on metaphase chromosomes and conventional interphase 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.

Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis

“…Paraformaldehyde-fixed cells (4%) were permeabilized for 30 min in 0.5% Triton X-100, and 3D-FISH was carried out as described previously (59). Glycerol treatment and flash-freezing in liquid nitrogen were omitted to preserve the structural integrity of the large syncytium of the myotube.…”

Chromosome positioning and the clustering of functionally related loci in yeast is driven by chromosomal interactions

“…Mammalian chromosomes reside in distinct regions of the nucleus termed chromosome territories. [15][16][17][18][19] These territories are not disjunct and a degree of chromosome mixing occurs. 18 So far, the existence of chromosome territories within the budding yeast nucleus is debated 20,21 although there is growing evidence of their existence.…”

Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos

“…Fluorescence in situ hybridization and signal detection FISH experiments were carried out essentially as described by Cremer et al 35 with modifications described in detail by Koehler et al 9,30 . Hybridization mixtures contained 50% formamide, 2x SSC, 10% dextran sulfate.…”