The authors acknowledge the technical assistance of the INGM Imaging Facility (Istituto Nazionale di Genetica Molecolare &#34;Romeo ed Enrica Invernizzi&#34; (INGM), Milan, Italy), in particular C. Cordiglieri, for assistance during 3D multicolor DNA FISH images acquisition. This work has been supported by the following grants to B.B.: EPIGEN Italian flagship program, Association Fran&#231;aise contre les Myopathies (AFM-Telethon, grant nr 18754) and Giovani Ricercatori, Italian Ministry of Health (GR-2011-02349383). This work has been supported by the following grant to F.M.: Fondazione Cariplo (Bando Giovani, grant nr 2018-0321).

1. DNA probe preparation and labelling procedures with nick translation
<ol>
	<li>Purify and clean bacterial artificial chromosomes (BACs), plasmids or PCR products with specific kits (Table of Materials), resuspend in ddH2O, check by electrophoresis on an agarose gel and quantify.</li>
	<li>Perform nick translation on 1.5&#8722;2 &#181;g of DNA from step 1.1 in a final volume of 50 &#181;L by mixing all the reagents in a 0.5 mL low binding DNA tube according to Table 1. 
	NOTE: Directly labelled probes can improve the signal to noise ratio. Kits to produce indirectly and directly labelled probes with various Alexa fluorochromes by nick translation are commercially available.</li>
	<li>Incubate the nick translation mix in a thermal mixer at 16 &#176;C for a time depending on the length of the starting DNA material: 45 min for PCR products (a pool of PCR products of 2,000 bp each) and up to 4 h for BAC DNA and plasmids.</li>
	<li>Check the size of the probes produced in step 1.3 by electrophoresis on a 2.2% agarose gel. 
	NOTE: The optimal probe size is &#60;200 bp (Figure 1A).
	<ol>
		<li>If the DNA is not digested enough, add 5 U of DNA polymerase I and 0.05 U of DNase I to the reaction and incubate for 1&#8722;2 h at 16 &#176;C and subsequently re-check. Stop the reaction with 0.5 mM EDTA (final concentration). Store probes at -20 &#176;C.</li>
	</ol>
	</li>
	<li>For each DNA FISH experiment, precipitate the following quantities of probes depending on the starting DNA material from which the probes are produced: 200 ng from nick translated PCR products, 100 ng from nick translated BACs, or 300 ng from nick translated plasmid. Add ddH2O up to 150 &#181;L, 20 &#181;g of unlabelled salmon sperm DNA, 3.5 &#181;g of species-specific Cot-1 DNA, 3 volumes of 100% EtOH, and 1/10 volume of 3 M sodium acetate pH 5.2. Precipitate at -80 &#176;C for 1 h.</li>
	<li>Centrifuge at maximum speed for 1 h at 4 &#176;C and discard the supernatant. Wash the pellet twice with 70% EtOH.</li>
	<li>Resuspend the pellet in 2 &#181;L of 100% formamide pH 7.0, shake at 40 &#176;C for 30 min (can take up to a few hours) and then add an equal volume of 4x saline-sodium citrate (SSC)/20% dextran sulfate.
	<ol>
		<li>Prepare 20x SSC by mixing 175.3 g of NaCl and 88.2 g of sodium citrate in a final volume of 1 L of H2O. Autoclave, filter and prepare aliquots. 
		NOTE: For multicolor DNA FISH, the different probes can be precipitated together except for probes that can anneal with one another. In these specific cases, treat them separately, precipitate and resuspend the probes dividing the reagents mentioned in steps 1.5&#8722;1.7 with respect to the number of probes. Pool the probes only after resuspension in formamide. If glass coverslips greater than 10 mm or less than 10 mm are used, scale up/down the volume of the reagents used in steps 1.5&#8722;1.7 proportionally to the slide size. Hybridization probes can be stored at -20 &#176;C for a long period of time (up to two months).</li>
	</ol>
	</li>
</ol>
2. Cell fixation, pre-treatment and permeabilization
NOTE: Permeabilization and deproteinization passages are crucial steps. The time of reaction and concentration of the reagents strongly depend on the cell type, the cytoplasm abundance, and the nuclear morphology.
<ol>
	<li>Cell fixation, pre-treatment and permeabilization for human primary T lymphocytes 
	NOTE: This protocol is suitable for small cells, with small nuclei and a low amount of cytoplasm.
	<ol>
		<li>Use glass coverslips (10 mm, thickness No. 1.5H).</li>
		<li>Wash the glass with ddH2O, then with 70% EtOH and let them dry. Use one glass for each well of a 24-well plate.</li>
		<li>Add 200 &#181;L of 0.1% poly-L-lysine (w/v) (Table of Materials) directly on the glass to form a drop. Pay attention as the drop must remain on the glass surface without touching the well for 2&#8722;5 min. Leave out the drop carefully, and let the glass dry for 30 min.</li>
		<li>Perform step 2.1.3 twice more.</li>
		<li>Put 200 &#181;L of suspension cells (2 x 106/mL, PBS suspension of ex vivo primary T lymphocytes) directly on the glass, allow the cells to seed at room temperature (RT) for 30 min.</li>
		<li>Quickly remove the drop. Add freshly made 4% PFA (prepared in PBS/0.1% TWEEN 20, pH 7.0, filtered) for 10 min and fix the seeded cells at RT.</li>
		<li>Wash three times for 5 min each with 0.05% Triton X-100/PBS (TPBS) at RT. Permeabilize with 0.5% TPBS for 10 min at RT.</li>
		<li>Perform RNase treatment by adding 2.5 &#181;L of RNase cocktail (Table of Materials) in 250 &#181;L of PBS/well in a 24-well plate for 1 h at 37 &#176;C.</li>
		<li>Rinse in PBS, add 20% glycerol/PBS, and incubate overnight (ON) at 4 &#176;C. 
		NOTE: This step can range from 1 h to ON. Slides can be kept in 20% glycerol/PBS at 4 &#176;C up to 7 days.</li>
		<li>Freeze on dry ice (15&#8722;30 s), thaw gradually at RT, and soak in 20% glycerol/PBS. Repeat step 2.1.9 another three times.</li>
		<li>Wash in 0.5% TPBS for 5 min at RT. Wash in 0.05% TPBS, twice for 5 min at RT. Incubate in 0.1 N HCl for 12 min at RT. Rinse in 2x SSC.</li>
		<li>Incubate in 50% formamide pH 7.0/2x SSC ON at RT. 
		NOTE: The incubation time can be optimized and eventually reduced. Slides can be kept in 50% formamide/2x SSC for several days.</li>
	</ol>
	</li>
	<li>Cell fixation, pre-treatment and permeabilization for human primary myoblasts 
	NOTE: This protocol is suitable for large cells, with a high amount of cytoplasm.
	<ol>
		<li>Grow human primary myoblasts directly on the coverslip glasses in 24-well plates in growth medium (Dulbecco&#39;s modified Eagle medium (DMEM), 20% fetal bovine serum (FBS), 25 ng/mL fibroblast growth factor (FGF), 10 ng/mL epidermal growth factor (EGF), 10 &#181;g/mL human insulin, 1x glutamine, 1x penicillin/streptomycin) for at least 24 h (reaching 50&#8722;70% of confluence). 
		NOTE: To facilitate the adhesion, gelatin or collagen or poly-L-lysine can be used to coat the coverslip prior the seeding.</li>
		<li>Rinse cells in 2&#8722;3 changes of PBS. Add freshly made 4% PFA and fix the cells for 10 min at RT.</li>
		<li>Wash three times for 3 min each in 0.01% TPBS at RT. Permeabilize in 0.5% TPBS for 10 min at RT.</li>
		<li>Perform RNase treatment by adding 2.5 &#181;L of RNase cocktail (Table of Materials) in 250 &#181;L of PBS/well in a 24-well plate for 1 h at 37 &#176;C.</li>
		<li>Rinse in PBS, add 20% glycerol/PBS, and incubate ON at RT. 
		NOTE: This step can range from 1 h to ON. Slides can be kept in 20% glycerol/PBS at 4 &#176;C up to 7 days.</li>
		<li>Freeze on dry ice (15&#8722;30 s), thaw gradually at RT, and soak in 20% glycerol/PBS. Repeat this step three times.</li>
		<li>Wash three times for 10 min each in PBS. Incubate in 0.1 N HCl for 5 min at RT. Rinse in 2x SSC.</li>
		<li>Incubate in 50% formamide pH 7.0/2x SSC ON at RT. 
		NOTE: The time of incubation can be optimized and eventually reduced. Slides can be kept in 50% formamide/2x SSC for several days.</li>
		<li>Equilibrate slides (kept in 50% formamide/2x SSC) in 2x SSC for 2 min. Then, equilibrate in PBS for 3 min.</li>
		<li>Treat with 0.01 N HCl/0.0025% pepsin from a few seconds up to 5 min, depending on the cell type. During this step, observe the cells under an optical microscope and stop the reaction (step 2.2.11) as soon as the nuclei are free from the cytoplasm, while maintaining their structure intact (e.g., nucleoli are still visible and intact).</li>
		<li>Inactivate the pepsin by washing twice for 5 min each in 50 mM MgCl2/PBS.</li>
		<li>Post-fix in 1% PFA/PBS for 1 min. Wash for 5 min in PBS. Wash twice for 5 min each in 2x SSC, and then add 50% formamide/2x SSC for at least 30 min.</li>
	</ol>
	</li>
</ol>
3. 3D multicolor DNA FISH hybridization
<ol>
	<li>Denature the probes at 80 &#176;C for 5 min, and then put quickly on ice.</li>
	<li>Load the hybridization probes on a clean microscope slide. Turn the coverslip with cells upside down on the drop of hybridization probes.</li>
	<li>Seal the coverslip with rubber cement. Let the rubber cement dry completely. Then place slides on a heating block and denature at 75 &#176;C for 4 min. 
	NOTE: The timing of denaturation and temperature of denaturation can be augmented, up to 80 &#176;C.</li>
	<li>Hybridize at 37 &#176;C ON in a metallic box floating in a water bath. 
	NOTE: To improve the signal to noise ratio, the hybridization temperature can go up to 42 &#176;C.</li>
	<li>Peel off rubber cement, immerse the slides in 2x SCC, strip off the glass coverslip and transfer it to 2x SSC in 6-well plate.</li>
	<li>Wash in 2x SSC three times for 5 min each at 37 &#176;C, shaking at 90 rpm in an incubator shaker. Wash in 0.1x SSC three times for 5 min each at 60 &#176;C, shaking at 90 rpm in an incubator shaker. Rinse briefly in 4x SSC/0.2% TWEEN 20.</li>
</ol>
4. 3D multicolor DNA FISH detection
NOTE: For directly labelled probes, skip steps 4.1 and 4.2.
<ol>
	<li>Block in 4x SSC/0.2% TWEEN 20/4% bovine serum albumin (BSA) for 20 min at 37 &#176;C in a 24-well plate, shaking at 20 rpm in an incubator shaker.</li>
	<li>Incubate with the appropriate concentration of anti-digoxygenin (1:150), and/or streptavidin (1:1,000) (Table of Materials) diluted in 4x SSC/0.2% TWEEN 20/4% BSA for 35 min in a dark and wet chamber at 37 &#176;C.</li>
	<li>Wash in 4x SSC/0.2% TWEEN 20 three times for 5 min each at 37 &#176;C, shaking at 90 rpm in a 6-well plate in an incubator shaker.</li>
	<li>Equilibrate in PBS and post-fix in 2% formaldehyde/PBS for 2 min at RT in a 24-well plate. 
	NOTE: Directly labelled probes do not need post-fixation.</li>
	<li>Wash 5x briefly in PBS. Stain with 1 ng/mL DAPI (4,6-diamidino-2-phenylindole)/PBS for 5 min at RT.</li>
	<li>Wash 5x briefly in PBS. Mount with antifade solution (Table of Materials).</li>
</ol>
5. 3D multicolor DNA FISH microscopy and analysis
<ol>
	<li>Acquire 3D images with a microscope system. 
	NOTE: Here, a widefield microscope with an axial distance of 0.2&#8722;0.25 &#956;m between consecutive sections (Figure 1B) is used.</li>
	<li>Analyze 3D image stacks using different software and tools (here, NuCL&#949;D or Nuclear Contacts Locator in 3D). 
	NOTE: The tool NuCL&#949;D has been developed in order to automatically analyze 3D multicolor DNA FISH in fluorescence cell image z-stacks. NuCL&#949;D reconstructs the nuclei from cell image stacks in 3D as well as detects and localizes fluorescent 3D spots. It measures the relative positioning of spots in the nucleus (e.g., distance from the centroid of the nuclei and/or periphery of the nuclei) and the maximum radius for each nucleus and inter-spots distances. The tool and the algorithm used are fully described in Cortesi et al.16.</li>
	<li>Analyze the data (e.g., 3D distances between specified genomic loci and the nuclear centroid and contact frequencies) retrieved by NuCL&#949;D. 
	NOTE: For 3D distances between specified genomic loci and the nuclear centroid, normalize distances on the maximum radius for each nucleus and represent these data as frequency distributions of normalized distances from the nuclear centroid. For long range interactions studies, contact frequencies are supposed to be in the range of 10&#8722;20%21 with a threshold inter-distance that can vary and can be put around 2 &#181;m35.
	<ol>
		<li>To perform statistical analysis, analyze approximately 100 nuclei per biological replicate. Represent 3D distances between specified genomic loci as cumulative frequency distributions of distances that are below the threshold inter-distance selected and use a t-test to assess the significance of differences in the distributions. Also, calculate the percentage of nuclei positive for the interactions that are below the threshold inter-distance selected, and use Fisher&#39;s exact test to assess the significance of differences in the percentages.</li>
	</ol>
	</li>
</ol>

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R., Guo, L., Lockett, S., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allele-specific+nuclear+positioning+of+the+monoallelically+expressed+astrocyte+marker+GFAP.">Allele-specific nuclear positioning of the monoallelically expressed astrocyte marker GFAP.</a> Genes &amp; Development. 22 (4), 489-498 (2008).</li><li>Kraus, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+structured+illumination+microscopy+of+nuclear+structures.">Quantitative 3D structured illumination microscopy of nuclear structures.</a> Nature Protocols. 12 (5), 1011-1028 (2017).</li><li>Solovei, I., Cremer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-FISH+on+cultured+cells+combined+with+immunostaining.">3D-FISH on cultured cells combined with immunostaining.</a> Methods in Molecular Biology. 659, 117-126 (2010).</li><li>Skinner, B. M., Johnson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+morphologies:+their+diversity+and+functional+relevance.">Nuclear morphologies: their diversity and functional relevance.</a> Chromosoma. 126 (2), 195-212 (2017).</li><li>Schoenfelder, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pluripotent+regulatory+circuitry+connecting+promoters+to+their+long-range+interacting+elements.">The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.</a> Genome Research. 25 (4), 582-597 (2015).</li><li>Lubeck, E., Cai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+systems+biology+by+super-resolution+imaging+and+combinatorial+labeling.">Single-cell systems biology by super-resolution imaging and combinatorial labeling.</a> Nature Methods. 9 (7), 743-748 (2012).</li><li>Beliveau, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+super-resolution+imaging+of+chromosomes+and+in+situ+haplotype+visualization+using+Oligopaint+FISH+probes.">Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes.</a> Nature Communication. 6, 7147 (2015).</li><li>Beliveau, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+design+and+synthesis+platform+for+visualizing+genomes+with+Oligopaint+FISH+probes.">Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes.</a> Proceedings of the National Academy of Sciences. 109 (52), 21301-21306 (2012).</li><li>Samacoits, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+framework+to+study+sub-cellular+RNA+localization.">A computational framework to study sub-cellular RNA localization.</a> Nature Communication. 9 (1), 4584 (2018).</li><li>Cardozo Gizzi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopy-Based+Chromosome+Conformation+Capture+Enables+Simultaneous+Visualization+of+Genome+Organization+and+Transcription+in+Intact+Organisms.">Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.</a> Molecular Cell. 74 (1), 212-222 (2019).</li><li>Mateo, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+DNA+folding+and+RNA+in+embryos+at+single-cell+resolution.">Visualizing DNA folding and RNA in embryos at single-cell resolution.</a> Nature. 568 (7750), 49-54 (2019).</li><li>Nir, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Walking+along+chromosomes+with+super-resolution+imaging,+contact+maps,+and+integrative+modeling.">Walking along chromosomes with super-resolution imaging, contact maps, and integrative modeling.</a> PLoS Genetics. 14 (12), 1007872 (2018).</li><li>Bintu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+chromatin+tracing+reveals+domains+and+cooperative+interactions+in+single+cells.">Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells.</a> Science. 362 (6413), (2018).</li></ol>

The authors have nothing to disclose.

The current method describes a step by step protocol to perform 3D multicolor DNA FISH on a wide range of human primary cells. Although DNA FISH is a technology in wide use, 3D multicolor DNA FISH on preserved 3D interphase nuclei is still difficult to perform in many laboratories, mainly due to the characteristics of the samples used23,24.
Probe nick translation is a fundamental step for successful 3D multicolor DNA FISH; many differe...

Higher eukaryotes need to systematically condense and compact a huge amount of genetic information in the minute 3D space of the nucleus1,2,3,4. Today, we know that the genome is spatially ordered in compartments and topologically associated domains5 and that the multiple levels of DNA folding generate contacts between different genomic regions that may involve chromatin loop formation6,7. The 3D dynamic looping of chromatin can influence many different biological processes such as transcription8,9, differentiation and development10,11, DNA repair12,13, while its perturbations are involved in various diseases14,15,16 and developmental defects15,17,18.
Many approaches have been developed to study the 3D genome organization. Chromosome conformation capture-based technologies (C-technologies, 3C, 4C, 5C, Hi-C and derivatives) have been developed to study genome organization in fixed cells3,4,19,20. Such approaches are based on the ability to capture the contact frequencies between genomic loci in physical proximity. C-technologies, depending on their complexity, catch the global 3D genome organization and nuclear topology of a cell population3,4,19,20. Nevertheless, 3D interactions are dynamic in time and space, highly variable between individual cells consisting of multiplex interactions, and are extensively heterogenous21,22.
3D multicolor DNA fluorescence in situ hybridization (FISH) is a technique that allows the visualization of specific genomic loci at a single cell level, enabling direct investigation of the 3D nuclear architecture in a complementary manner to C-technologies. It represents a technology currently used to unambiguously validate C-results. 3D multicolor DNA FISH uses fluorescently labeled probes complementary to the genomic loci of interests. The use of different fluorophores and suitable microscopy equipment allow contemporary visualization of multiple targets within the nuclear space23,24. In recent years, FISH has been combined with technological advances in microscopy to obtain the visualization of fine-scale structures at high resolution25,26 or with CRISPR-Cas approaches for the visualization of the nucleic acids in live imaging27,28. Despite wide adoption, the 3D multicolor DNA FISH approach is still considered difficult in many laboratories because the biological material used must be adapted.
Here, we provide a comprehensive protocol for 3D multicolor DNA FISH (from cell/probe preparation to data analysis) applicable to a wide range of human primary cells, enabling the visualization of multiple genomic loci and preserving the 3D structure of nuclei. In order to study nuclear architecture, the 3D structure of nuclei must be preserved. For this reason, contrasting from other existing protocols29,30,31, we avoid the use of an alcohol gradient and the storage of the coverslips in alcohol that can affect chromatin structure32. The method is adapted from preserved 3D DNA FISH protocols24,33 to be applied to a wide range of human primary cells, both isolated ex vivo or cultured in vitro. There are permeabilization and deproteinization parameters for different nuclear morphology and cytological characteristics (e.g., different degrees of nuclear compaction, cytoskeleton abundance)34. These parameters are often generally described in other protocols24,33, without providing a clear discrimination of the procedure within different cell types. Furthermore, we developed a specific tool named NuCL&#949;D (nuclear contacts locator in 3D)16, providing principles for data analysis that will improve the 3D proximity between different loci and their nuclear topological distribution within the nuclear space in an automated way.

<table><tbody><tr><td>24-well plates</td><td>Thermo Fisher Scientific</td><td>142475</td><td></td></tr><tr><td>6-well plates</td><td>Thermo Fisher Scientific</td><td>140675</td><td></td></tr><tr><td>Anti-Digoxigenin 488</td><td>DBA</td><td>DI7488</td><td></td></tr><tr><td>b-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>bFGF</td><td>PeproTech</td><td>100-18B</td><td></td></tr><tr><td>Biotin 11 d-UTP</td><td>Thermo Fisher Scientific</td><td>R0081</td><td></td></tr><tr><td>BSA (bovine serum albumine)</td><td>Sigma</td><td>A7030</td><td></td></tr><tr><td>Coverlsips</td><td>Marienfeld</td><td>117500</td><td></td></tr><tr><td>CY3 d-UTP</td><td>GE Healthcare</td><td>PA53022</td><td></td></tr><tr><td>DAPI (4,6-diamidino-2-phenylindole)</td><td>Thermo Fisher Scientific</td><td>D21490</td><td></td></tr><tr><td>Deoxyribonucleic acids single strand from salmon testes</td><td>Sigma</td><td>D7656</td><td></td></tr><tr><td>Dextran sulfate (powder)</td><td>Santa Cruz</td><td>sc-203917A</td><td></td></tr><tr><td>Digoxigenin 11 d-UTP</td><td>Roche</td><td>11093088910</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>21969-035 500mL</td><td></td></tr><tr><td>DNA polymerase I</td><td>Thermo Fisher Scientific</td><td>18010-017</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>AMPD1</td><td></td></tr><tr><td>dNTPs (C-G-A-T)</td><td>Euroclone</td><td>BL0423A/C/G</td><td></td></tr><tr><td>EGF</td><td>Sigma</td><td>E9644.2MG</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>02860-1L</td><td></td></tr><tr><td>FBS Hyclone</td><td>Thermo Fisher Scientific</td><td>SH30109</td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma</td><td>F8775-25mL</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>Glutammine</td><td>Thermo Fisher Scientific</td><td>25030-024 100mL</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516-100mL</td><td></td></tr><tr><td>Glycogen</td><td>Thermo Fisher Scientific</td><td>AM9510</td><td></td></tr><tr><td>HCl</td><td>Sigma</td><td>30721</td><td></td></tr><tr><td>Human &lt;em&gt;Cot-1&lt;/em&gt; DNA</td><td>Thermo Fisher Scientific</td><td>15279-001</td><td></td></tr><tr><td>Insulin Human</td><td>Sigma</td><td>I9278-5 mL</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>63069</td><td></td></tr><tr><td>NaAc (Sodium Acetate, pH 5.2, 3 M)</td><td>Sigma</td><td>S2889</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127-25G</td><td></td></tr><tr><td>PBS (phosphate-buffered saline)</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>Pennycillin/Streptavidin</td><td>Thermo Fisher Scientific</td><td>15070-063 100mL</td><td></td></tr><tr><td>Pepsin</td><td>Biorad</td><td>P6887</td><td></td></tr><tr><td>PhasePrep BAC DNA Kit</td><td>Sigma</td><td>NA0100-1KT</td><td></td></tr><tr><td>Poly-L-lysine solution</td><td>Sigma</td><td>P8920</td><td></td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Thermo Fisher Scientific</td><td>P36970</td><td></td></tr><tr><td>PureLink Quick Gel Extraction &amp;amp; PCR Purification Combo Kit</td><td>Thermo Fisher Scientific</td><td>K220001</td><td></td></tr><tr><td>PureLink Quick Plasmid Miniprep Kit</td><td>Thermo Fisher Scientific</td><td>K210010</td><td></td></tr><tr><td>RNAse cocktail</td><td>Thermo Fisher Scientific</td><td>AM2288</td><td></td></tr><tr><td>Rubbercement</td><td>Bostik</td><td></td><td></td></tr><tr><td>Slides</td><td>VWR</td><td>631-0114</td><td></td></tr><tr><td>Streptavidina Alexa fluor 647</td><td>Thermo Fisher Scientific</td><td>S21374</td><td></td></tr><tr><td>Tri-Sodium Citrate</td><td>Sigma</td><td>1110379026</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>T3253-500g</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-250mL</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma</td><td>P9416-100mL</td><td></td></tr></tbody></table>

3d multicolor dna fish tool to study nuclear architecture in human primary cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

The method of 3D multicolor DNA FISH described in this article allows contemporary visualization of different genomic loci within preserved 3D nuclei (Figure 1B). This protocol permits the measurement of distances between alleles, and different genomic loci in order to evaluate their spatial proximity, and to assess their location within the nuclear space (e.g., loci distance from the centroid or the periphery of the nuclei)16. However, there are many...

Watch this Scientific Journal Video about 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells at JoVE.com

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

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Research

JoVE Journal

Genetics

10.2K Views.  Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", INGM. 3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

Video: 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Medicine

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Immunology and Infection

Analysis Workflow of Nuclear Protein Staining in Human Primary Cells

A Versatile Pipeline for Analyzing Dynamic Changes in Nuclear Bodies in a Variety of Cell Types

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the immunofluorescence technique. Investigating the dynamics of these foci under diverse cellular conditions via microscopy yields valuable insights into the molecular mechanisms governing cellular identity and functions. However, performing immunofluorescence assays across different cell types and assessing alterations in the assembly, diffusion, and distribution of these foci present numerous challenges. These challenges encompass complexities in sample preparation, determination of parameters for analyzing imaging data, and management of substantial data volumes. Moreover, existing imaging workflows are often tailored for proficient users, thereby limiting accessibility to a broader audience.
In this study, we introduce an optimized immunofluorescence protocol tailored for investigating nuclear proteins in different human primary T cell types that can be customized for any protein of interest and cell type. Furthermore, we present a method for unbiasedly quantifying protein staining, whether they form distinct foci or exhibit a diffuse nuclear distribution.
Our proposed method offers a comprehensive guide, from cellular staining to analysis, leveraging a semi-automated pipeline developed in Jython and executable in Fiji. Furthermore, we provide a user-friendly Python script to streamline data management, publicly accessible on a Google Colab notebook. Our approach has demonstrated efficacy in yielding highly informative immunofluorescence analyses for proteins with diverse patterns of nuclear organization across different&#160;contexts.

Author Spotlight: Comprehensive Epigenetic Analysis for Investigating Human Cellular Plasticity and Environmental Adaptation Using Immunofluorescence Assays

This method describes an immunofluorescence protocol and quantification pipeline for evaluating protein distribution with varied nuclear organization patterns in human T lymphocytes. This protocol provides step-by-step guidance, starting from sample preparation and continuing through the execution of semi-automated analysis in Fiji, concluding with data handling by a Google Colab notebook.

Various nuclear processes, such as transcriptional control, occur within discrete structures known as foci that are discernable through the ...

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Developmental Biology

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Biology

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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.

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 ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

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