Name: combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization
Uploaded: 2013-02-03T14:00:00
Description: Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

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 &amp; 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).

1. DNA Probe Labeling with Fluorophores: Nick Translation (~ 6 hr)
<ol>
 <li>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.</li>
 <li>Incubate DNA in RNase A for 30 min at 37 &deg;C (All reagents are listed in Table 1).</li>
 <li>Incubate the nick translation reaction for 2 hr at 16 &deg;C (see Tables 2 and 3</strong...

<ol>
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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...

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 specific interactions/associations of DNA sequences and/or proteins within the nucleus. Furthermore, while genome-wide high throughput techniques such as chromatin immunoprecipitation (ChIP-seq) or chromosome capture conformation coupled with deep sequencing (4C-seq, 5C, Hi-C) provide global data on cell populations12, immunofluorescence/DNA FISH techniques enable analyses at the single cell level.
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-FISH). The advantage of this protocol is the combined visualization of DNA and preservation of protein structures. Our experience in this field has enabled us to improve and simplify existing protocols. Although we have used this protocol to detect DNA double-stranded breaks in lymphocytes undergoing recombination, this method can be applied to other proteins and other cell-types.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>H2O</td>
 <td>Fisher</td>
 <td># BP2470</td>
 <td></td>
 </tr>
 <tr>
 <td>RNase A</td>
 <td>Sigma</td>
 <td># R4642</td>
 <td></td>
 </tr>
 <tr>
 <td>dNTP</td>
 <td>Sigma</td>
 <td># DNTP100</td>
 <td></td>
 </tr>
 <tr>
 <td>Alexa dUTP</td>
 <td>Invitrogen</td>
 <td># C11397 to C-11401</td>
 <td></td>
 </tr>
 <tr>
 <td>Cy3 or Cy5 dUTP</td>
 <td>Fisher</td>
 <td># 45-001-xxx</td>
 <td></td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Roche</td>
 <td># 04536282001</td>
 <td></td>
 </tr>
 <tr>
 <td>DNA Pol I</td>
 <td>Biolabs</td>
 <td># M0209</td>
 <td></td>
 </tr>
 <tr>
 <td>0.025 &mu;m filters</td>
 <td>Millipore</td>
 <td># VSWP02500</td>
 <td></td>
 </tr>
 <tr>
 <td>Cot-1 DNA 1 mg/ml</td>
 <td>Invitrogen</td>
 <td># 18440</td>
 <td></td>
 </tr>
 <tr>
 <td>Hybloc DNA 1 mg/ml</td>
 <td>Applied Genetics</td>
 <td># MHB</td>
 <td></td>
 </tr>
 <tr>
 <td>Salmon sperm</td>
 <td>Sigma</td>
 <td># D1626</td>
 <td>powder to be resuspended at 10 mg/ml in H2O</td>
 </tr>
 <tr>
 <td>NaAc (Sodium Acetate, pH 5.2, buffer solution)</td>
 <td>Sigma</td>
 <td># S7899</td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll 400 (Mol Biol grade)</td>
 <td>Fisher</td>
 <td># 525</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyvinylpyrrolidone (Mol Biol grade)</td>
 <td>Fisher</td>
 <td># BP431</td>
 <td></td>
 </tr>
 <tr>
 <td>Dextran sulfate powder</td>
 <td>Sigma</td>
 <td># D8906</td>
 <td></td>
 </tr>
 <tr>
 <td>SSPE (Saline-Sodium Phosphate-EDTA) 20x solution</td>
 <td>Fisher</td>
 <td># BP1328</td>
 <td></td>
 </tr>
 <tr>
 <td>Formamide</td>
 <td>Fisher</td>
 <td># BP227</td>
 <td></td>
 </tr>
 <tr>
 <td>Coverslips</td>
 <td>Fisher</td>
 <td># 12-548-B</td>
 <td></td>
 </tr>
 <tr>
 <td>Slides</td>
 <td>Fisher</td>
 <td># 12-550</td>
 <td></td>
 </tr>
 <tr>
 <td>6-well plates</td>
 <td>Fisher</td>
 <td># 0720080</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS, 10x</td>
 <td>Fisher</td>
 <td># MT-46-013-CM</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-lysine solution</td>
 <td>Sigma</td>
 <td># P8920</td>
 <td></td>
 </tr>
 <tr>
 <td>Paraformaldehyde, prills, 95%</td>
 <td>Sigma</td>
 <td># 441244</td>
 <td></td>
 </tr>
 <tr>
 <td>Triton-X-100, Mol Biol grade</td>
 <td>Sigma</td>
 <td># T8787</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA (Bovine Serum Albumin) Fraction V</td>
 <td>Fisher</td>
 <td># BP 1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Normal goat serum</td>
 <td>Vector Labs</td>
 <td># S-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween-20, Mol Biol grade</td>
 <td>Sigma</td>
 <td># P9416</td>
 <td></td>
 </tr>
 <tr>
 <td>SSC (Saline Sodium Citrate) 20x solution</td>
 <td>Fisher</td>
 <td># BP1325</td>
 <td></td>
 </tr>
 <tr>
 <td>ProLong Gold antifade reagent</td>
 <td>Invitrogen</td>
 <td># P36930</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI (4',6-diamidino-2-phenylindole)</td>
 <td>Sigma</td>
 <td># D9542</td>
 <td></td>
 </tr>
 <tr>
 <td>Best test one coat rubber cement</td>
 <td>Art or office supply stores</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. Specific reagents and small equipment.</td>
 </tr>
 </tbody>
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combined immunofluorescence and dna fish on 3d-preserved interphase nuclei to study changes in 3d nuclear organization

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.

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...

Watch this Scientific Journal Video about Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization at JoVE.com

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

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 ...

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Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

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