Understanding how the genome is organized within the cell nucleus is increasingly recognized to be important to understand gene regulation. In 3D DNA fluorescence in situ hybridization (3D DNA FISH) labeled probes complementary to specific loci of interest are hybridized to the genome. The samples are then imaged using fluorescence microscopy, collecting z-stacks through the nuclei, and the relative positions of the hybridized probes are analyzed in the reconstructed 3D images. In this way 3D DNA FISH provides a powerful tool to interrogate how the organization of specific genomic loci changes in response to stimuli. This chapter describes protocols which have allowed us to produce consistent data in cultured cells and paraffin-embedded tissue sections.
Keywords: Chromatin; DNA; FISH; Gene regulation; Genetics; Nuclear organization.