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). Alternative methods of direct labeling may be used (Example: FISH Tag, Invitrogen).</li>
 <li>Inactivate the reaction for one hr at -80 &deg;C or overnight at -20 &deg;C.</li>
 <li>Determine probe size on a 2% agarose gel. The smear should be between 100 and 1,000 bp, with the majority at approximately 300-500 bp (Note that the smear of Cy5-probes is not visible). If the DNA is not digested enough, more DNA Pol I and DNase I can be added to the reaction and incubated for two more hr at 16 &deg;C.</li>
 <li>Purify probes on 0.025 mm filters in two liter beakers filled with H20 for two hr in the dark.</li>
 <li>Add blocking factors to the probes as follows: 10 &mu;g of Cot-1 DNA, 10 &mu;g of Hybloc DNA and 100 &mu;g of salmon sperm for 10 &mu;g of DNA probes. Store DNA probes at -20 &deg;C.</li>
</ol>
2. Probe Precipitation/Denaturation/Pre-annealing (3-4 hr)
<ol>
 <li>Between 0.3 &mu;g and 1 &mu;g of DNA probe is used per coverslip. Mix probes with 0.1 volume of 3M NaAc (pH 5.2) and 2.5 volumes of 100% ethanol, incubate for one hr at -80 &deg;C or overnight at -20 &deg;C and spin down at 13,000 rpm at 4 &deg;C for 30 min. Note that the amount of DNA probe may be adjusted depending on the quality of the DNA and the nuclear region of hybridization. Alternative methods of direct labeling may allow the use of reduced amounts of probe. For detection of multiple DNA FISH signals on one slide, the different probes can be precipitated together (except probes that should not be pre-annealed, like repeat sequences). Probes for several experiments performed at the same time can also be precipitated together. Pellets should be lightly colored according to the fluorophores used (if small amounts of probes are precipitated, the pellet may not be visible).</li>
 <li>Dry pellets and resuspend them in 15 &mu;l hybridization buffer (see Table 4) per coverslip with shaking (using an Eppendorf thermomixer) in the dark at 37-45 &deg;C (for approximately 20 min).</li>
 <li>Denature probes for 5 min at 75-95 &deg;C.</li>
 <li>Pre-anneal probes at 37 &deg;C for 30-60 min. Length of pre-annealing may be adjusted depending on the complexity and quality of the probes.</li>
 <li>Apply probes to the coverslips for overnight hybridization (see Section 3).</li>
</ol>
3. 3-dimensional DNA FISH - First Day (~ 3 hr)
<ol>
 <li>Non-adherent cells are concentrated in a small volume of 1x PBS (~ 2x105 cells in 5-10 &mu;l 1x PBS per coverslip) and dropped on the center of a poly-L-lysine coated coverslip. Adherent cells should be cultured on coverslips for at least 24 hr (~ 70% confluence) (coverslips may be coated with 0.1% gelatin). Square 18x18 to 24-24 mm coverslips are placed in 6-well plates, and throughout the protocol, two ml of each solution is used per well (alternatively round 1.5 mm coverslips can be placed in 12/24-well plates).</li>
 <li>Fix cells in 2% paraformaldehyde / 1x PBS, pH 7-7.4, for 10 min at RT (4% PFA stocks can be aliquoted, stored at -20 &deg;C). Paraformaldehyde can be purchased in solution (16%), or 4% stock solutions can be prepared from powder/prills. Paraformaldehyde powder is dissolved in 1x PBS at &gt; 60 &deg;C (pH may be increased to 8-9 to facilitate dissolution), cooled-down on ice, adjusted to pH 7-7.4, filtered, aliquoted in falcons and stored for weeks at -20 &deg;C. </li>
 <li>After three rinses in 1x PBS, permeabilize cells in iced-cold 0.4% Triton-X-100 / 1x PBS for 5 min on ice. Length of permeabilization may be adjusted depending on the cell-type. N.B.: If DNA FISH is combined with immunofluorescence, the immunostaining should be performed at this stage (see Section 4).</li>
 <li>After three rinses in 1x PBS, incubate cells with 0.1 &mu;g/&mu;l RNase A in 1x PBS for one hr at 37 &deg;C. Coverslips are placed cell-side down onto a drop of 100 &mu;l of solution on parafilm or a slide in a humid chamber. Coverslips are then carefully removed with forceps. If any resistance is encountered, coverslips should be flooded with 1x PBS in order to avoid damaging the cells.</li>
 <li>After three rinses in 1x PBS, permeabilize cells in iced-cold 0.7% Triton-X-100 / 0.1 M HCl for 10 min on ice.</li>
 <li>After three rinses in 1x PBS, denature cells in 1.9 M HCl for 30 min at RT. Alternately, cells can be denatured in 50% formamide / 2x SSC at 75-80 &deg;C for at least 30 min. Note that hybridization length (and temperature) may be optimized depending on the cell-type.</li>
 <li>After three rinses in ice-cold 1x PBS, hybridize cells with probes overnight at 37 &deg;C in a dark and humid chamber. Coverslips are placed cell-side down onto a drop of 15 &mu;l of probe on a slide, and sealed with rubber cement.</li>
</ol>
4. 3-dimensional Immuno-FISH - First Day (~ 6-7 hr)
<ol>
 <li>For combined immunofluorescence /DNA FISH, immuno-staining should be performed after the permeabilization in 0.4% Triton-X-100 (Step 3.3).</li>
 <li>Incubate cells in blocking solution for 30 min at RT (2.5% bovine serum albumin (BSA), 10% Normal Goat Serum, 0.1% Tween-20). The blocking solution is filtered, aliquoted in 1.5 ml Eppendorfs and stored for months at -20 &deg;C.</li>
 <li>Incubate cells with the primary antibody raised against the protein or the histone modification of interest diluted in blocking solution, for one hr at RT in a humid chamber. Here we show the example of an antibody against phosphorylated serine-139 of H2AX (&gamma;-H2AX, Millipore; See Table 5 for details). Coverslips are placed cell-side down onto a drop of 100 &mu;l of solution on parafilm or a slide in a humid chamber. Coverslips are then carefully removed with forceps (flooded with 1x PBS if necessary).Note that dilution and incubation length may be adjusted depending on the quality of the antibody and cell-types.</li>
 <li>Wash cells in 0.2% BSA / 0.1% Tween-20 / 1x PBS, three times 5 min at RT with shaking.</li>
 <li>Incubate cells with the appropriate fluorophore-conjugated secondary antibody diluted in blocking solution for one hr at RT in a dark and humid chamber. Here we show an example of detection with a secondary goat anti-mouse antibody (Alexa Fluor 488 or 555, Invitrogen; See Table 5 for details). Hapten-conjugated secondary antibodies (biotin, digoxigenin, etc) could also be used. In this case, an extra step for appropriate detection (streptavidin, anti-dig, etc) can be performed either before or after over-night DNA-FISH hybridization.</li>
 <li>Rinse cells in 0.1% Tween-20 / 1x PBS, three times 5 min at RT with shaking in the dark.</li>
 <li>Post-fix cells in 2% paraformaldehyde / 1x PBS for 10 min at RT in the dark.</li>
 <li>Continue DNA FISH as above from incubation in RNase A (Step 3.4).</li>
</ol>
5. 3-dimensional DNA FISH / Immuno-FISH - Second Day (~ 2 hr)
<ol>
 <li>Carefully remove rubber cement, flood coverslips with 2x SSC, carefully remove with forceps and place them in wells containing washing solution.</li>
 <li>Wash cells in 2x SSC for 30 min at 37 &deg;C in the dark with shaking.</li>
 <li>Wash cells in 2x SSC for 30 min at RT in the dark with shaking.</li>
 <li>Wash cells in 1x SSC for 30 min at RT in the dark with shaking. Note that in case of denaturation with formamide, washes should be replaced by the following: 3 washes in 50% formamide / 2x SSC and 3 washes in 2x SSC, 5 min each, at 37 &deg;C.</li>
 <li>Mount cells in ProLong Gold mounting medium containing 1.5 &mu;g/ml DAPI (4,6-diamidino-2-phenylindole) for counterstaining of total DNA. Coverslips are placed cell-side down onto a drop of 10-15 &mu;l of mounting medium on a slide and sealed with nail polish. Slides are kept for months at -20 &deg;C.</li>
</ol>
6. 3D Microscopy and Analysis
<ol>
 <li>3D images can be acquired with different microscope systems. In our experiments, optical sections separated by 0.3 &mu;m are collected on a Leica SP5 AOBS confocal system (Acousto-Optical Beam Splitter). Note that the separation between optical planes may be adjusted depending on the type of analysis.</li>
 <li>3D image stacks can be analyzed using different software and tools. We use the Image J software to assess distance measurements between loci of interest and to analyze different types of association of the loci of interest with sub-nuclear compartments or proteins.</li>
 <li>All statistical analyses are performed to compare two (or more) different biological conditions with pairwise assessment(s) of significance: comparison between different cell types or stages, comparison between different loci of interest. In all statistical tests, P-values &le; 5.00E-2 (a = 0.05) are taken to be significant (1.00E-2 &le; P &le; 5.00E-2 * significant; 1.00E-3 &le; P &le; 1.00E-2 ** very significant; P &lt; 1.00E-3 *** highly significant).</li>
</ol>
Statistical analysis of the entire distributions of distances between two alleles. We first construct cumulative distribution frequency curves over the entire range of measured distances between the two alleles.To assess the significance of differences in the distributions of empirical inter-allelic distances we use the nonparametric two-sample Kolmogorov-Smirnov (KS) test 13,14 .
Statistical analysis of close association (i.e. pairing) of two alleles using an optimal distance cut-off. To identify the range of most robust differences in distances between two alleles or loci, we use a series of two-tail Fisher exact test 15 s. Namely, at each measured distance we test whether one condition is significantly over-represented at shorter distances between the two samples. The minimum in the distribution of P-Values indicates the range of distances that should be considered as a cut-off for close association (i.e. pairing) of the two alleles. Subsequently the two-tail Fisher exact test is used to assess the significance of pairing of two alleles at the identified cut-off value15. Multiple testing corrections are applied to account for the total number of Fisher tests performed 16.
Statistical analysis of association of the locus of interest with sub-nuclear compartments or proteins. The two-tail Fisher-exact test is used to analyze the significance of association of the locus of interest with different compartments or proteins 15.

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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=Differences+in+the+localization+and+morphology+of+chromosomes+in+the+human+nucleus.">Differences in the localization and morphology of chromosomes in the human nucleus.</a> The Journal of cell biology. 145, 1119-1131 (1999).</li><li>Chaumeil, J., Le Baccon, P., Wutz, A., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced.">A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced.</a> Genes Dev. 20, 2223-2237 (2006).</li><li>Walter, 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=Towards+many+colors+in+FISH+on+3D-preserved+interphase+nuclei.">Towards many colors in FISH on 3D-preserved interphase nuclei.</a> Cytogenetic and genome research. 114, 367-378 (2006).</li><li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annual review of cell and developmental biology. 26, 285-314 (2010).</li><li>Schermelleh, L., Heintzmann, R., Leonhardt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+super-resolution+fluorescence+microscopy.">A guide to super-resolution fluorescence microscopy.</a> The Journal of cell biology. 190, 165-175 (2010).</li><li>Dobbie, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMX:+a+new+platform+for+multimodal,+multichannel+wide-field+imaging.">OMX: a new platform for multimodal, multichannel wide-field imaging.</a> Cold Spring Harbor protocols. , 899-909 (2011).</li><li>Boyle, S., Rodesch, M. J., Halvensleben, H. A., Jeddeloh, J. A., Bickmore, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+with+high-complexity+repeat-free+oligonucleotide+probes+generated+by+massively+parallel+synthesis.">Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis.</a> Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 19, 901-909 (2011).</li></ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O</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 &amp;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 H&lt;sub&gt;2&lt;/sub&gt;O</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&#039;,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 colspan="4">&lt;strong&gt;Table 1. &lt;/strong&gt; Specific reagents and small equipment.</td></tr></tbody></table>

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 ...

combined-immunofluorescence-dna-fish-on-3d-preserved-interphase

Research

JoVE Journal

Biology

18.3K Views.  New York 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).

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

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetics

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Cancer Research

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.

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 ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Developmental Biology

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 ...

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 ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of the most common hematological malignancies. The identifying characteristic of MM is accumulated malignant plasma cells in bone marrow. FISH reports for MM mainly focus on purified or identified clonal plasma cells, rather than all nucleated cells, by sorting with anti-CD138 magnetic beads or marking with cytoplasmic immunoglobulin light chain &#954; or &#955;. Bone marrow interphase nuclei are usually obtained from fresh bone marrow cells. However, satisfactory enrichment of plasma cell specimens requires large amounts of fresh heparin anti-coagulated bone marrow, which cannot be obtained in the case of difficult bone marrow extraction or a bone marrow dry tap. Herein, we establish a novel method to improve the success of FISH detection on stained or unstained bone marrow smears. Bone marrow smears are easier to obtain than anticoagulated bone marrow specimens.

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Here, we present a protocol for improving the success of interphase fluorescence in situ hybridization detection on bone marrow smears from multiple myeloma patients.

Fluorescence in situ hybridization (FISH) detection is an indispensable method in genetic risk stratification in multiple myeloma (MM), which is one of ...

Medicine

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

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

Summary

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Chapters in this video

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

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

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

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

Robust 3D DNA FISH Using Directly Labeled Probes

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Interphase Fluorescence in situ Hybridization of Bone Marrow Smears of Multiple Myeloma

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes