Name: Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization (Video) | JoVE Journal
Uploaded: 2013-02-03T14:00:00.000+00:00
Duration: 13 min 55 s
Description: Learn to combine immunofluorescence and DNA FISH for studying 3D nuclear organization in interphase nuclei. This protocol enables simultaneous detection of histone modifications and specific DNA sequences using confocal microscopy.

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5 minutes

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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<li>Chaumeil, J., Le Baccon, P., Wutz, A., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+role+for+Xist+RNA+in+the+formation+of+a+repressive+nuclear+compartment+into+which+genes+are+recruited+when+silenced%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">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/pubmed?term=((Towards+many+colors+in+FISH+on+3D-preserved+interphase+nuclei%5BTitle%5D)+AND+%22Cytogenetic+and+genome+research%22%5BJournal%5D)">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/pubmed?term=((A+new+wave+of+cellular+imaging%5BTitle%5D)+AND+%22Annual+review+of+cell+and+developmental+biology%22%5BJournal%5D)">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/pubmed?term=((A+guide+to+super-resolution+fluorescence+microscopy%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">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/pubmed?term=((OMX%3A+a+new+platform+for+multimodal%2C+multichannel+wide-field+imaging%5BTitle%5D)+AND+%22Cold+Spring+Harbor+protocols%22%5BJournal%5D)">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/pubmed?term=((Fluorescence+in+situ+hybridization+with+high-complexity+repeat-free+oligonucleotide+probes+generated+by+massively+parallel+synthesis%5BTitle%5D)+AND+%22Chromosome+research+%3A+an+international+journal+on+the+molecular%2C+supramolecular+and+evolutionary+aspects+of+chromosome+biology%22%5BJournal%5D)">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

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...

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 ...

The overall goal of this procedure is to detect chromatin modifications and DNA sequences simultaneously using 3D confocal microscopy first specific DNA probes are generated. Meanwhile, the experimental cells are fixed perme and immuno stained. For the histone markers of interest.The cells are then denatured and an overnight fluorescence inside you. Hybridization or fish is performed using one or more DNA probes. Finally, the cells are washed and mounted for 3D fluorescence.Microscopy analysis of the resulting images reveals nuclear localization of loci of interest and association with specific his stone modifications. The main advantage of this technique is that it combines visualization of nuclear protein, including stones with genomic loci in 3D preserve nuclei, and can help answer key questions in the field of chromatin epigenetic nuclear organization or gene regulation. For this procedure, either backs plasmids or PCR products can be used for labeling.These should be prepared as described in the accompanying text. Note that for a robust fish signal, the probes should spend at least 10 kilobase pair. Begin this procedure by combining one microgram of DNA and 0.17 micrograms of RNAs A in a micro centrifuge tube.Incubate for 30 minutes at 37 degrees Celsius. Following the incubation, briefly inactivate the RNAs A on ice and add the appropriate amount of Nick translation. Mix incubate for two hours at 16 degrees Celsius in a refrigerated water bath or a cool block to inactivate the reaction.Place the tubes at negative 80 degrees Celsius for one hour or negative 20 degrees Celsius overnight. Next, determine the probe size on a 2%agros gel. The smear should be between 101, 000 base pair with the majority at approximately 300 to 500 base pair.Note that the smear of sci-fi probes is not visible. Once the size has been determined, purify the probes by dialysis on 0.02. Five millimeter filters in two liter beakers filled with water.Gently pipette 200 micrograms of probe onto the center of each floating filter, then incubate for two hours in the dark. Following the incubation, use a pipette to gently transfer the probe from the filters to a fresh micro centrifuge tube. Next, add blocking factors to the probes as follows.One microgram of CO 1D NA.One microgram of high block DNA and 10 micrograms of salmon sperm for one microgram of DNA probe and mix store the DNA probes at negative 20 degrees Celsius. On the day of the fish procedure, the probes are precipitated and prepared for use. Note that the steps described in this section of the video should be performed in parallel with the steps in the following section.To precipitate the probes, follow them and combine them with 0.1 volume of three molar sodium acetate at pH 5.2 and 2.5 volumes of 100%Ethanol incubate for one hour at negative 80 degrees celsius. After the incubation, spin the probes down at 16, 000 times G at four degrees Celsius for 30 minutes. Following the spin, discard the supernatant and air dry the pellets in the dark.Depending on the amount and color of the probes, the pellet may or may not be visible. Resus suspend the pellet in 15 microliters of hybridization buffer per test condition and shake using an einor thermo mixer in the dark at 37 to 45 degrees Celsius for approximately 20 minutes. Next to denature the probes incubate them for five minutes at 75 to 95 degrees Celsius.Then to pre probes, incubate them at 37 degrees Celsius for 30 to 60 minutes on the thermo mixer. Immediately following the incubation, the probes will be applied to cells on cover slips as described in the next section of the video. Note that for the following steps, incubation times should be modified as necessary for the antibodies and cells used.The steps described in this section of the video should be performed in parallel with the steps in the previous section as described in the accompanying text. Drop non-adherent cells on top poly lysine coated cover slips in wells of six well plates and let them attach for five minutes. Then gently add two milliliters of 2%paraform aldehyde in PBS incubate for 10 minutes at room temperature following the incubation.Rinse each well with PB S3 times then to permeable the cells, add two milliliters of ice cold, 0.4%Triton X 100 in PBS and incubate for five minutes on ice. After aspirating the permeable solution if DNA fish will be combined with immunofluorescence, proceed as follows. First pipette 100 microliters of blocking solution onto param in a humidified chamber.Place the cover slip cell side down onto the blocking solution and incubate for 30 minutes at room temperature. Next, transfer. The cover slips to 100 microliter drops of the primary antibody diluted in blocking solution.Here the antibody used recognizes phosphorylated Syrian 1 39 of H two A x or gamma H two A x. Incubate the slides in a humidified chamber for one hour at room temperature. After the incubation, use forceps to carefully remove the cover slips.If the cover slips are difficult to pick up, flood with PBS and try again. Transfer each slide to a well of a six. Well plate containing two milliliters of 0.2%BSA 0.1%tween 20 in PBS wash the cover slips in this solution three times for five minutes at room temperature with shaking, then incubate cells with the appropriate fluorophore conjugated secondary antibody diluted in blocking solution for one hour at room temperature in a dark and humid chamber on param.As before following the incubation, rinse the cells in 0.1%tween 20 in PB S3 times for five minutes at room temperature in the dark to postfix the cells, submerge them in two milliliters of 2%para formaldehyde in PBS for 10 minutes at room temperature in the dark. Once the cells have been stained or if they are not stained, continue DNA fish by rinsing the cover slips three times in PBS. Then place the cover slips cell side down onto a 100 microliter drop of 0.1 milligrams per milliliter.RNAs on param in a humidified chamber and incubate for one hour at 37 degrees Celsius. After the incubation, rinse the cover slips three times in PBS then perme the cells in ice cold 0.7%Trix 100 in 0.1 molar hydrochloric acid for 10 minutes on ice in the dark. After three rinses in PBS denature the cells in 1.9 molar hydrochloric acid for 30 minutes at room temperature in the dark and wash three more times in ice cold PBS next to hybridize cells with the probes.Place the cover slips cell side down onto a 15 microliter drop of probe prepared in the previous section of this video on a microscope slide, seal the slide with rubber cement and incubate overnight at 37 degrees Celsius in a dark humidified chamber. The next day, use forceps to carefully remove the rubber cement from the slides. Then flu the cover slips with two Acts SSC and use forceps to carefully remove the cover slips with the cells.Then place them in wells containing washing solution. Next, perform the following washes with shaking in the dark two XSSC for 30 minutes at 37 degrees Celsius, two XSSC for 30 minutes at room temperature and one XSSC for 30 minutes at room temperature to mount the slides, place the cover slips cell side down onto a 10 to 15 microliter drop of prolonged gold mounting medium containing 1.5 milligrams per milliliter DPI seal with nail polish. The slides can be stored for months at negative 20 degrees Celsius.Once the slides are ready, collect 3D images with optical sections separated by 0.3 microns and analyze as described in the accompanying document to determine whether changes in association of the two alleles of the antigen receptor loci are linked with the regulation of the process of VDJ recombination during lymphocyte development, wild type and mutant cells deficient in the recombinase protein rag one were used to examine a locus of interest using a DNA fish probe generated from a back that spans approximately 200 kilobase pair encompassing the genomic region. Distances between alleles were measured between the center of mass of each back signal in 3D image stacks dashed circles, outline nucleus shapes. The scale bars shown are one micron cumulative frequency distribution curves were then generated to show the entire range of distances between the two alleles measured in the wild type and mutant cells.The non-parametric two sample KS test was then used to assess the significance of the differences between the two distributions. The range of the most robust differences in inter allelic distances was assessed using a series of two tail fisher exact tests indicated by the blue dots. The most robust P values indicated by the higher dots were found for the distances around one micron.Thus, the cutoff for close association or pairing of the two alleles was set up at one micron. Cumulative frequency curves shown here indicate the range of distances for close association of the two alleles from zero to 1.5 microns in the two different biological conditions. The significance of pairing of the two alleles was assessed using the two tail fisher exact test for a cutoff at one micron.This graph shows that in the mutant condition, the two alleles are not associated with high frequency as in the wild type. Thus, the missing protein rag one is involved inic pairing. After watching this video, you should have a good understanding of how to combine immunofluorescence and DNA fish.This technique paved the way for researchers by giving insights into the global changes in nuclear architecture, the behavior of specific genomic loci and regions within the nuclear space and the association of specific nuclear proteins within these regions. And it helps understanding the mechanisms underlying different disease following this procedure. Other methods like genome wide ice throughput techniques such as chromatin, immuno precipitations, or chips seek or chromosome capture confirmation coupled with deep sequencing like fore five C or IC, can be performed to provide global data on cell populations.

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

Research

JoVE Journal

Biology

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

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.

FISH for Pre-implantation Genetic Diagnosis 

In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins γH2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

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

Robust 3D DNA FISH Using Directly Labeled Probes

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).

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

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals.  Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

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.

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

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.

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

Learn to combine immunofluorescence and DNA FISH for studying 3D nuclear organization in interphase nuclei. This protocol enables simultaneous detection of histone modifications and specific DNA sequences using confocal microscopy.

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

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