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.<br ...

<ol>
	<li>Cremer, T., Cremer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+territories,+nuclear+architecture+and+gene+regulation+in+mammalian+cells.">Chromosome territories, nuclear architecture and gene regulation in mammalian cells.</a> Nature Reviews: Genetics. 2 (4), 292-301 (2001).</li><li>Lieberman-Aiden, E., 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=Comprehensive+mapping+of+long-range+interactions+reveals+folding+principles+of+the+human+genome.">Comprehensive mapping of long-range interactions reveals folding principles of the human genome.</a> Science. 326 (5950), 289-293 (2009).</li><li>Schmitt, A. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 Cot-1 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>MgCl2</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 &#38; 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 ...

3D multicolor DNA FISH enables the visualization of multiple genomic loci within preserved nuclei to ambiguously define their reciprocal interaction and localization at single cell level. 3D multicolor DNA FISH allows the direct investigation of the nuclear architecture. In general, it works in conjunction with chromosome capture-based technologies making the technique an available tool for C data validation within single cells.Demonstrating the procedure will be Federica Marasca. She's a postdoc in my laboratory. Begin by incubating the nick translation mix in a thermal mixer at 16 degrees Celsius according to the length of the starting DNA material.At the end of the incubation, check the size of the probes on a 2.2%agarose gel. For each DNA FISH experiment, precipitate the appropriate quantity of probe according to the starting DNA material from which the probes were produced in 150 microliters of double distilled water, 20 micrograms of unlabeled salmon sperm DNA, 3.5 micrograms of species specific Cot-1 DNA, three volumes of 100%ethanol, and a 1/10th volume of three molar sodium acetate for one hour at minus 80 degrees Celsius. At the end of the incubation, centrifuge the sample at maximum speed for one hour at four degrees Celsius.After discarding the supernatant, wash the pellet two times with 500 microliters of 70%ethanol. After the second wash, resuspend the pellet in two microliters of 100%formamide and shake the probe for 30 minutes at 40 degrees Celsius before adding an equal volume of 4X saline sodium citrate in 20%dextran sulfate. For the fixation pre-treatment and permeabilization of small cells with small nuclei and a low amount of cytoplasm, first add two times 10 to the sixth cells in 200 microliters of PBS directly onto glass coverslips in individual wells of a 24-well plate and allow the cells to settle for 30 minutes at room temperature.At the end of the incubation, quickly replace the PBS with 300 microliters of freshly prepared 4%paraformaldehyde supplemented with 0.1%Tween 20. After 10 minutes, wash the fixed cells three times in 300 microliters of 0.05%TPBS for five minutes per wash at room temperature. After the last wash, permeabilize the T cells with 300 microliters of 0.5%TPBS for 10 minutes at room temperature before treating the cells with 250 microliters per well of RNAse cocktail diluted 1:100 in PBS for one hour at 37 degrees Celsius.At the end of the incubation, rinse the cells in 300 microliters of PBS and add 300 microliters of 20%glycerol in PBS to each well with an overnight incubation at four degrees Celsius. The next morning, place the glass on dry ice for 15 to 30 seconds to freeze the cells before gradually thawing the samples at room temperature and soaking them in 300 microliters of 20%glycerol in PBS for 30 seconds. After freeze/thawing the cells three more times as just demonstrated, wash the samples in 300 microliters of 0.5%TPBS for five minutes at room temperature, followed by two washes in 300 microliters of 0.05%TPBS for five minutes per wash at room temperature.After the last wash, treat the cells with 300 microliters of 0.1 normal hydrochloric acid for 12 minutes at room temperature, followed by two rinses in 300 microliters of saline sodium citrate. Then fix the samples overnight in 300 microliters of 50%formamide in 2X saline sodium citrate at room temperature. For the fixation, pre-treatment and permeabilization of large cells with a high amount of cytoplasm, fix, pre-treat and permeabilize the cells similarly to as demonstrated for human primary T cells and treat the samples with 300 microliters of 0.0025%pepsin in 0.01 normal hydrochloric acid for a few seconds up to five minutes.Observe the cells under an optical microscope and stop the reaction with two five-minute washes in 300 microliters of 50 millimolar magnesium chloride as soon as the nuclei are free from the cytoplasm but the nucleoli are still visible and intact. For 3D multicolor DNA FISH, denature the hybridization probes at 80 degrees Celsius for five minutes and immediately place the probes on ice. Load the probes onto a clean microscope slide and place one coverslip of cells onto each drop of probe.Seal the coverslip edges with rubber cement. When the cement has dried completely, denature the samples on a 75 degrees Celsius heating block. After four minutes, hydridize the samples at 37 degrees Celsius overnight in a metallic box floating in a water bath.The next morning, peel off the rubber cement and with the slides immersed in 2X saline sodium citrate carefully lift off the coverslips. Place each coverslip into individual wells of a six-well plate containing two milliliters of fresh saline sodium citrate per well for two five-minute washes at 37 degrees Celsius with shaking at 90 revolutions per minute. At the end of the incubation, rinse the coverslips briefly in two milliliters of 0.2%Tween 20 in 4X saline sodium citrate.For 3D multicolor DNA FISH detection, transfer the coverslips into individual wells of a new 24-well plate and block any nonspecific binding in 300 microliters of blocking buffer for 20 minutes at 37 degrees Celsius and 20 revolutions per minute. At the end of the incubation, treat the samples with the appropriate concentration of anti-digoxigenin and/or streptavidin diluted in blocking buffer for 35 minutes in a dark and wet chamber at 37 degrees Celsius. At the end of the incubation, transfer the samples to individual wells of a six-well plate and wash the samples three times in two milliliters of 0.2%Tween 20 and 4X saline sodium citrate for five minutes per wash with shaking at 90 revolutions per minute.After the last wash, equilibrate the samples in two milliliters of PBS before transferring the coverslips to a new 24-well plate for post-fixing in 300 microliters of 2%formaldehyde in PBS per well for two minutes at room temperature. Wash the fixed cells five times briefly in 300 microliters of PBS, followed by staining with DAPI diluted at one nanogram per milliliter in 300 microliters of PBS for five minutes at room temperature. Wash the coverslips five more times in PBS and mount the samples with an appropriate mounting medium.Then acquire 3D images with a microscope system. 3D multicolor DNA FISH allows the contemporary visualization of different genomic loci within preserved 3D nuclei. For DNA probe preparation, a probe size of less than 200 base pairs ensures a successful 3D multicolor DNA FISH procedure.Suboptimal DNA FISH probes produced by nick translation can be partially or over digested. Over digested probes will result in a nonspecific signal due to a loss of specificity in the hybridization and a consequent increase of the background. For human primary T lymphocytes and small cells with small nuclei and a low amount of cytoplasm, a 12-minute 0.1 normal hydrochloric acid treatment is recommended to promote nuclei accessibility to the DNA probes and to preserve the nuclear integrity.Cytoplasmic pepsin digestion is not needed to obtain a DNA FISH good signal in small cells. For human primary myoblasts and cells that have large nuclei and abundant cytoplasm, however, the pepsinization step is fundamental. A short and suboptimal cytoskeleton pepsinization will hamper the entry of the probe into the nuclei ending in the absence of a DNA FISH signal.However, if the cells are over pepsinized, the nuclei will not remain intact losing their 3D structure. A successful 3D multicolor DNA FISH will result in the presence of signal within the nuclei. I suggest working with fresh biological material, testing probes before performing the 3D multicolor DNA FISH, preparing fresh solutions and being precise with incubation times and the temperatures.Remember that formamide and HCL are hazardous substances and should always be used in a chemical fume hood with appropriate disposable materials.

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Genetics

10.1K Views.  Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", INGM. 3D multicolor DNA FISH enables the visualization of multiple genomic loci within preserved nuclei to ambiguously define their reciprocal interaction and localization at single cell level. 3D multicolor DNA FISH allows the direct investigation of the nuclear architecture. In general, it works in conjunction with chromosome capture-based technologies making the technique an available tool for C data validation within single cells.Demonstrating the procedure will be Federica Marasca. She'...