title: The Relationship Between Human Nucleolar Organizer Regions and Nucleoli, Probed by 3D-ImmunoFISH.
abstract: 3D-immunoFISH is a valuable technique to compare the localization of DNA sequences and proteins in cells where three-dimensional structure has been preserved. As nucleoli contain a multitude of protein factors dedicated to ribosome biogenesis and form around specific chromosomal loci, 3D-immunoFISH is a particularly relevant technique for their study. In human cells, nucleoli form around transcriptionally active ribosomal gene (rDNA) arrays termed nucleolar organizer regions (NORs) positioned on the p-arms of each of the acrocentric chromosomes. Here, we provide a protocol for fixing and permeabilizing human cells grown on microscope slides such that nucleolar proteins can be visualized using antibodies and NORs visualized by DNA FISH. Antibodies against UBF recognize transcriptionally active rDNA/NORs and NOP52 antibodies provide a convenient way of visualizing the nucleolar volume. We describe a probe designed to visualize rDNA and introduce a probe comprised of NOR distal sequences, which can be used to identify or count individual NORs.
authors: Centre for Chromosome Biology, National University of Ireland, Bioscience Research Building, University Road, Galway, Ireland.
authors: Marjolein
authors: M
authors: van Sluis
authors: Centre for Chromosome Biology, National University of Ireland, Bioscience Research Building, University Road, Galway, Ireland.
authors: Chelly
authors: C
authors: van Vuuren
authors: Centre for Chromosome Biology, National University of Ireland, Bioscience Research Building, University Road, Galway, Ireland. brian.mcstay@nuigalway.ie.
authors: Brian
authors: B
authors: McStay
mesh_terms: D002466:Cell Nucleolus / Q000235:genetics* / Q000378:metabolism; D002875:Chromosomes; D015342:DNA Probes; D004275:DNA, Ribosomal / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D056426:Genetic Loci; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009697:Nucleolus Organizer Region / Q000235:genetics* / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D015342:DNA Probes; D004275:DNA, Ribosomal
keywords: 3D-immunoFISH; Human acrocentric chromosomes; Nucleolar organizer region (NOR); Nucleolus; Ribosomal genes