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Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Published: February 3rd, 2013



1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Epigenetic mechanisms trigger establishment and inheritance of developmental and cell-type specific transcriptional profiles. At one level this involves modulation of chromatin packaging that defines active or silent genomic regions. On a larger scale, global 3D organization of the genome and nuclear architecture also play a role in the control of transcriptional patterns. Thus, dissection of these epigenomic features is essential for a full understanding of how genes are regulated 6-11.

Combined immunofluorescence and 3D DNA FISH provide a unique opportunity to complement molecular and biochemical analyses by assessing ....

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1. DNA Probe Labeling with Fluorophores: Nick Translation (~ 6 hr)

  1. Clean BAC DNA (prepared by maxi-prep) or plasmids or PCR products, all resuspended in H2O, can be used for labeling. Note that for a robust FISH signal, probes should span at least 10 kb.
  2. Incubate DNA in RNase A for 30 min at 37 °C (All reagents are listed in Table 1).
  3. Incubate the nick translation reaction for 2 hr at 16 °C (see Tables 2 and 3

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DNA and immuno-FISH are used in the Skok lab to study the changes in nuclear organization associated with the process of V(D)J recombination of antigen receptor loci during B and T lymphocyte development. The techniques detailed above enable us to i) measure distances between the two ends of a locus (contraction) ii) measure distances between alleles or loci (pairing), iii) analyze the DNA damage occurring within loci, iv) assess the location of alleles and loci relative to nuclear sub-compartments (repressive per.......

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The techniques detailed above were used in our lab to analyze the regulation of V(D)J recombination of the Immunoglobulin and Tcra/d loci in developing lymphocytes 30,31 . We are confident that this technique can be adapted for detection of various nuclear proteins, nuclear compartments and loci, in different cell-types. Modifications of the protocol may be necessary, and in this case the major steps to focus on are the following. First, the length of permeabilization can be adjusted de.......

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We would like to thank the members of the Skok lab, especially Susannah Hewitt, for discussions and comments. This work is supported by the National Institute of Health grants R01GM086852, RC1CA145746 (J.A.S.). J.A.S. is a Leukemia & Lymphoma Society scholar. J.C. is an Irvington Institute Fellow of the Cancer Research Institute. M.M. is supported by a National Science Foundation Grant Integrative Graduate Education and Research Traineeship (NSF IGERT 0333389).


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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalogue Number Comments
H2O Fisher # BP2470
RNase A Sigma # R4642
dNTP Sigma # DNTP100
Alexa dUTP Invitrogen # C11397 to C-11401
Cy3 or Cy5 dUTP Fisher # 45-001-xxx
DNase I Roche # 04536282001
DNA Pol I Biolabs # M0209
0.025 μm filters Millipore # VSWP02500
Cot-1 DNA 1 mg/ml Invitrogen # 18440
Hybloc DNA 1 mg/ml Applied Genetics # MHB
Salmon sperm Sigma # D1626 powder to be resuspended at 10 mg/ml in H2O
NaAc (Sodium Acetate, pH 5.2, buffer solution) Sigma # S7899
Ficoll 400 (Mol Biol grade) Fisher # 525
Polyvinylpyrrolidone (Mol Biol grade) Fisher # BP431
Dextran sulfate powder Sigma # D8906
SSPE (Saline-Sodium Phosphate-EDTA) 20x solution Fisher # BP1328
Formamide Fisher # BP227
Coverslips Fisher # 12-548-B
Slides Fisher # 12-550
6-well plates Fisher # 0720080
PBS, 10x Fisher # MT-46-013-CM
Poly-L-lysine solution Sigma # P8920
Paraformaldehyde, prills, 95% Sigma # 441244
Triton-X-100, Mol Biol grade Sigma # T8787
BSA (Bovine Serum Albumin) Fraction V Fisher # BP 1600
Normal goat serum Vector Labs # S-1000
Tween-20, Mol Biol grade Sigma # P9416
SSC (Saline Sodium Citrate) 20x solution Fisher # BP1325
ProLong Gold antifade reagent Invitrogen # P36930
DAPI (4',6-diamidino-2-phenylindole) Sigma # D9542
Best test one coat rubber cement Art or office supply stores
Table 1. Specific reagents and small equipment.

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