The authors thank all previous and present members of the department of Reproduction and Development of the Erasmus MC for helpful and stimulating discussions during the past years. We also want to thank the three anonymous reviewers for providing helpful feedback. Work in the Gribnau lab is supported by funding from the Dutch research council (NWO-VICI) and an ERC starting grant.

1. Induction of Differentiation in Female Embryonic Stem Cells to Induce X Chromosome Inactivation
NOTE: Female mouse embryonic stem cells (available upon request) are grown under standard ES cell conditions on gelatinized culture dishes coated with mouse embryonic fibroblasts (MEFs). We here assume that the reader is familiar with standard cell culture techniques39-41. To induce differentiation, ES cells grown in T25 dishes will be separated from the MEFs, and will be plated in differentiation medium.
<ol>
	<li>Remove ES medium from the ES cell culture, and wash twice with cell culture PBS.</li>
	<li>Add 1.5 ml trypsin-EDTA (pre-warmed to 37 &#176;C), and incubate cells at 37 &#176;C for 7 min. After 3.5 min, shake the dish gently to break up the colonies.</li>
	<li>Inactivate the trypsin-EDTA by adding 3.5 ml of differentiation medium to the cells. Pipette cells up and down to obtain a single cell solution, and collect in a 15 ml tube. Spin for 5 min at 200 x g, and collect cells in 10 ml of differentiation medium.</li>
	<li>Add cell suspension to a non-gelatinized culture dish, and incubate for 1 hr in a cell culture incubator. NOTE: MEFs will be able to adhere to the non-gelatinized culture dish, whereas ES cells will remain in suspension.</li>
	<li>In the meantime, add sterilized glass coverslips (24 x 24 mm) to 6-well plates, add 0.2% gelatin, and incubate for minimal 5 min at room temperature.</li>
	<li>After 1 hr, collect the supernatant of the pre-plated ES cell culture, which will mainly consist of non-adhering ES cells. Plate ES cells to 6-well plates containing coverslips. NOTE: Use &#8537;th of a confluent T25 dish for 6 wells of a 6-well plate. This will allow a confluent differentiation well after 3 days of differentiation. Change medium on a daily basis. For longer differentiation time courses, plate pre-plated ES cells in differentiation medium in bacterial dishes, and incubate on a cell shaker integrated into the cell culture incubator. This will allow the cells to form embryoid bodies, which can be plated to coverslips 1-2 days prior to fixation.</li>
</ol>
2. Fixation of Differentiated ES Cells for Subsequent DNA-RNA FISH Analysis
<ol>
	<li>Remove differentiation medium from differentiation cultures, and wash twice with cell culture PBS. NOTE: Add PBS carefully to prevent the cells being washed away.</li>
	<li>Remove cell culture PBS and fix cells by adding 4% paraformaldehyde (PFA)/PBS, and incubate for 10 min at room temperature. CAUTION: PFA is toxic and can cause significant health risk when being inhaled or when in contact to skin or eyes. Furthermore, PFA is carcinogenic.</li>
	<li>Remove 4% PFA/PBS, and wash coverslips 3x with 70% ethanol. NOTE: Correct washing is important, as any trace of PFA will interfere with subsequent FISH steps. Coverslips can be stored in 70% ethanol at -20 &#176;C for several months prior to FISH analysis.</li>
</ol>
3. Labeling of DNA Probes for FISH Experiments
NOTE: Obtain a DNA fragment of the gene of interest (minimal length &#62;2 kb for RNA FISH, or &#62;20 kb for DNA FISH), or a BAC covering the gene of interest. For RNA detection, a smaller probe can be sufficient compared to DNA detection, since most likely transcription will result in multiple transcripts, which can be simultaneously detected. For a strong signal on the single copy DNA strand, a longer probe is required, to be able to detect a sufficient number of incorporated haptens. Preferably, a non-transcribed genomic region is chosen for designing the DNA probe. In addition, when using a smaller probe to detect the RNA, it will be prevented that the genomic loci from which the RNA is transcribed is being detected in the combined DNA-RNA FISH approach, although this cannot be fully excluded. To detect mouse Xist, a 5.5 kb cDNA probe was used. For detection of the X chromosome, several BAC probes can be used. Good results have been obtained with a mixture of BAC RP23-100E1 and BAC CT7-474E4, which are located in close proximity to the Xist locus. Depending on the gene or chromosome of interest, several different probes might be required to obtain optimal results. Whenever working with materials which will be used for RNA-FISH, use sterile filter tips, RNAse free H2O, and work in a clean environment.
<ol>
	<li>Take 1 &#181;g of DNA and dilute in H2O in a total volume of 16 &#181;l. Add 4 &#181;l of digoxigenin or biotin labeling solution (see reagents), mix by vortexing and incubate at 16 &#176;C for 90 min. NOTE: The biotin or digoxigenin-labeled nucleotides will now be incorporated by nick-translation.</li>
	<li>In the meantime, prepare G50 columns to remove non-integrated nucleotides. Take a 2 ml syringe, remove the stamper, and add sterilized cotton into the syringe. Add G50 balls in solution to fill the syringe, place syringe in a 15 ml tube and centrifuge at 642 x g for 1 min. NOTE: Around 80% of the syringe should now be filled with G50. Repeat when less G50 is present.</li>
	<li>After 90 min, add 40 &#181;l of H2O to the nick-translation reaction mixture, and add the reaction to the G50 column. Add an empty tube under the column, and centrifuge at 642 x g for 2 min. Collect the flow through in a reaction tube.</li>
	<li>Measure the volume of the flow through with a pipette, and add H2O to reach a total volume of 200 &#181;l. Precipitate the flow through by adding 13.3 &#181;l salmon sperm DNA (10 mg/ml), 13.3 &#181;l yeast tRNA (10 mg/ml), 26.7 &#181;l mouse Cot-1 DNA (1 mg/ml) and 28 &#181;l 2 M NaAc, pH 5.6. Mix by vortexing, and add 533 &#181;l ice cold 100% ethanol. Shake the tube, and centrifuge at 16,200 x g for 30 min at 4 &#176;C.</li>
	<li>Remove the supernatant, and wash pellet twice with 70% ethanol. Centrifuge at 16,200 x g for 5 min at 4 &#176;C, and air dry the pellet. Add 50 &#181;l of 50+ hybridization solution, and incubate at 37 &#176;C for 30 min to facilitate dissolving. NOTE: Labeled probe can be stored for several years at -20 &#176;C. CAUTION: 50+ hybridization solution contains formamide, which is toxic, can irritate skin, eyes and the respiratory system, and is also a teratogen.</li>
</ol>
4. Permeabilization, Pre-treatment and Hybridization of Fixed Cells for Combined DNA-RNA FISH
<ol>
	<li>Rehydrate fixed cells on coverslips, by removing 70% ethanol and the addition of cell culture PBS to the 6-well plates. Incubate for 5 min. Repeat in total three times.</li>
	<li>Remove cell culture PBS, and add 0.2% pepsin to the 6-well plates, to permeabilize the cells. Incubate 6-well plates in a water bath at 37 &#176;C for 4 min. Remove pepsin, and inactivate with RNAse free H2O.</li>
	<li>Post-fix cells by incubating with 4% PFA/PBS for 5 min at room temperature. After fixation, wash twice with cell culture PBS for 5 min.</li>
	<li>Dehydrate cells by incubating with 70% ethanol for 3 min, 90% ethanol for 3 min and 100% ethanol for 3 min. Transfer coverslips out of 6-well plates, and air dry coverslips for several minutes.</li>
	<li>Age cells by incubating coverslips at 65 &#176;C for 1 hr on a heat plate.</li>
	<li>Pre-hybridize FISH probe with mouse Cot-1 DNA by adding together for every coverslip to be analyzed, 25 &#181;l of 50+ hybridization solution, 0.5 &#181;l mouse Cot-1 DNA, 1 &#181;l of biotin-labeled Xist probe and 1 &#181;l of digoxigenin labeled BAC probe detecting the X chromosome. Mix by vortexing, and denature at 99 &#176;C for 5 min. Incubate immediately at 37 &#176;C for 45 min, to allow Cot-1 DNA to hybridize to repeat sequences present in the probes.</li>
	<li>After aging, spot 50 &#181;l of denaturation buffer (consisting of 70% formamide/2x SSC/10 mM phosphate buffer) on a glass slide, and put coverslips on top with cells facing towards the denaturation buffer. Incubate at 65 &#176;C for 2 min.</li>
	<li>Add coverslips back to 6-well plates, and post-fix cells by incubating in ice cold 70% ethanol for 5 min.</li>
	<li>Dehydrate cells by subsequent incubation in 70% ethanol for 3 min, 90% ethanol for 3 min and 100% ethanol for 3 min. Remove coverslips from 6-well plates, and let air dry for several minutes.</li>
	<li>Spot pre-hybridized probe on a glass slide, and incubate coverslip on top. Place slides in a humidified chamber filled with 50 ml 50% formamide/2x SSC, and incubate overnight at 37 &#176;C.</li>
</ol>
5. Post-hybridization Washes and Antibody Mediated Detection of Probe
<ol>
	<li>Fill 6-well plates with 2x SSC, and pre-warm till 42 &#176;C. Add coverslips after overnight incubation, and incubate at 42 &#176;C for 5 min.</li>
	<li>Remove 2x SSC, and incubate coverslips with 50% formamide/2x SSC for 10 min at 42 &#176;C. Repeat in total 3 times (30 min of washing altogether).</li>
	<li>Remove 50% formamide/2x SSC, and wash slides with Tris-saline-Tween (TST) for 2 x 5 min at room temperature.</li>
	<li>Spot 50 &#181;l Tris-saline-BSA (TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for blocking during 30 min at room temperature in a TST-humidified dark chamber.</li>
	<li>Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.</li>
	<li>Spot 50 &#181;l of sheep-anti-dig antibody (1:500 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside-down for the first incubation step during 30 min at room temperature in a TST-humidified dark chamber.</li>
	<li>Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.</li>
	<li>Spot 50 &#181;l of rabbit-anti-sheep antibody conjugated with FITC (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the second incubation step during 30 min at room temperature in a TST-humidified dark chamber.</li>
	<li>Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.</li>
	<li>Spot 50 &#181;l of goat-anti-rabbit antibody conjugated with FITC (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the third incubation step during 30 min at room temperature in a TST-humidified dark chamber.</li>
	<li>Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.</li>
	<li>Spot 50 &#181;l of mouse-anti-biotin antibody (1:200 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the fourth incubation step during 30 min at room temperature in a TST-humidified dark chamber.</li>
	<li>Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.</li>
	<li>Spot 50 &#181;l of donkey-anti-mouse antibody conjugated with Rhodamine (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the fifth incubation step during 30 min at room temperature in a TST-humidified dark chamber.</li>
	<li>Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST.</li>
	<li>Spot 50 &#181;l of goat-anti-horse antibody conjugated with Rhodamine (1:250 in TSBSA) per coverslip on new glass slides, and incubate coverslips upside down for the sixth incubation step during 30 min at room temperature in a TST-humidified dark chamber. NOTE: the goat-anti-horse antibody will recognize the donkey-anti-mouse antibody; when available to the researcher, a goat-anti-donkey antibody will work as well.</li>
	<li>Transfer coverslips back to 6-well plates, and wash 2 x 5 min with TST. Wash additional 5 min with Tris-saline (TS).</li>
	<li>Dehydrate cells by subsequent incubation in 70% ethanol for 3 min, 90% ethanol for 3 min and 100% ethanol for 3 min. Remove coverslips from 6-well plates, and let air dry for several minutes.</li>
	<li>Spot a drop of mounting medium with DAPI (see reagents) on a glass slide, and add coverslip upside down on top. Seal slides with nail varnish and assess FISH results by fluorescent microscopy (Figure 1).</li>
</ol>

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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=Simultaneous+in+situ+detection+of+RNA,+DNA,+and+protein+using+tyramide-coupled+immunofluorescence.">Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence.</a> Methods Mol Biol. 292, 215-230 (2005).</li><li>Pollex, T., 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=Live-Cell+Imaging+Combined+with+Immunofluorescence,+RNA,+or+DNA+FISH+to+Study+the+Nuclear+Dynamics+and+Expression+of+the+X-Inactivation+Center.">Live-Cell Imaging Combined with Immunofluorescence, RNA, or DNA FISH to Study the Nuclear Dynamics and Expression of the X-Inactivation Center.</a> Methods Mol Biol. 1042, 13-31 (2013).</li><li>Chaumeil, 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=Combined+immunofluorescence,+RNA+fluorescent+in+situ+hybridization,+and+DNA+fluorescent+in+situ+hybridization+to+study+chromatin+changes,+transcriptional+activity,+nuclear+organization,+and+X-chromosome+inactivation.">Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.</a> Methods Mol Biol. 463, 297-308 (2008).</li><li>Lin, S., Talbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+mouse+and+human+embryonic+stem+cells.">Methods for culturing mouse and human embryonic stem cells.</a> Methods Mol Biol. 690, 31-56 (2011).</li><li>Meissner, 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=Derivation+and+manipulation+of+murine+embryonic+stem+cells.">Derivation and manipulation of murine embryonic stem cells.</a> Methods Mol Biol. 482, 3-19 (2009).</li><li>Nichols, J., Ying, Q. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+propagation+of+embryonic+stem+cells+in+serum-+and+feeder-free+culture.">Derivation and propagation of embryonic stem cells in serum- and feeder-free culture.</a> Methods Mol Biol. 329, 91-98 (2006).</li></ol>

The authors have nothing to disclose.

Combined DNA-RNA FISH can be technically challenging, as conditions suitable for DNA FISH can be too harsh for less stable RNA molecules. Several approaches have been applied to study both DNA and RNA in the same cell, circumventing the need for simultaneous incubation with probes detecting both the DNA and RNA. For example, in a superimposition approach, first RNA FISH is performed, and cells are imaged, and coordinates are taken. Subsequently, the same slides are used for DNA FISH, during which the signal of RNA FISH is lost. After DNA FISH, the cells are again imaged, and the pictures obtained from both the RNA and DNA FISH are superimposed. Although this approach will work in almost all cases, as the DNA of fixed cells is stable, and is not affected by the treatment needed for initial RNA FISH, this is a very laborious approach which will cost at least four days. In another approach, first the RNA FISH is performed, the RNA FISH signal is fixed by addition of fixatives, after which DNA FISH is applied. Although also this approach can work, some of the fluorescent signal derived from the RNA FISH can be lost upon fixation. This might especially hinder the detection of transcripts which are only expressed at a low level. The herein presented approach is optimized for the simultaneous detection of both one RNA target and one DNA target in a single FISH experiment, and has the advantage that both results can be obtained in a single experiment within two days, with reliable results.
Several steps in the FISH protocol are crucial to obtain optimal results. First, whenever dealing with RNA molecules, one should be careful not to introduce RNAse in any buffer or solution used. Hence, a clean working environment should be established, and filter tips should be used when pipetting reagents being used for FISH. Care should be taken to use RNAse free compounds (for example RNAse free water, cell culture grade PBS, absolutely pure ethanol etc.). When these simple rules are followed, it is generally not necessary to use additional RNAse inhibitors supplemented to the reagents used. Second, the permeabilization procedure using pepsin is a gentle equilibrium between too little, or too much permeabilization. Depending on the particular cell type used, it might be required to optimize the time allowed for permeabilization, or the pepsin concentrations. The latter also depends on your particular batch of pepsin, as the activity might vary. As an alternative, permeabilization using 0.1% Triton-X100/PBS for 5 min results in good results under certain conditions, and has the advantage that FISH using this method of permeabilization can be combined with immunostaining to detect nuclear or cytoplasmic proteins. As a third important aspect of FISH, the aging, denaturation, hybridization and washing temperatures should be kept constant. Even small deviations from the target temperature can result in less efficient denaturation, hybridization, or less stringent washes, which will result in more background staining. As an alternative to the 1 hr aging step at 65 &#176;C followed by denaturation at 65 &#176;C for 2 min, also an initial denaturation at 80 &#176;C for 5 min, without aging, can give good results. We prefer the aging procedure, as in general this procedure gives more reliable DNA FISH signals. Lastly, certain DNA probes will be so specific, that they will not require three layers of antibodies for sufficient detection. This should be empirically tested for every newly generated probe.
Although the current protocol works robustly in the study of X chromosome inactivation and detection of Xist, combined detection of RNA and DNA might be more complicated in other settings. This might especially be the case when the RNA species which is aimed to be detected is only expressed at very low levels, is full of repeat sequences or is a relatively short transcript. In these situations, it might be rather difficult to design an optimal probe, which will allow the efficient hybridization to a limited, not optimal available amount of target. In such situations, it might be worth optimizing first conditions for RNA FISH. Several alternative probes could be tried, and the duration of probe labeling by Nick-translation could be titrated, to optimize the amount of incorporated haptens. Importantly, it should be verified that the aimed RNA is expressed in the cells to be investigated, by for example verifying expression by RT-PCR. Also when analyzing a different type of cells, it is essential to include cells in which expression has previously been detected as positive controls, as this might allow the distinction between a technical, FISH related problem, or the simple absence of expression in the new type of cells analyzed. When, despite verified expression, the use of various probes and optimization of labeling conditions, RNA FISH does not result in a clear signal, it might be worth changing the hybridization conditions, by varying the amount of formamide used, or the duration and temperature of probe hybridization. Alternatively, directly labeled oligonucleotide probes might be used9-10. The same optimization steps can subsequently be applied to improve DNA FISH, and the simultaneous detection of DNA and RNA.
Using the combined DNA-RNA FISH protocol, we have been able to study X chromosome inactivation in differentiating female ES cells. Similar results have been obtained in our studies using different cell types and embryos, and we are currently further optimizing conditions to apply combined DNA-RNA FISH on tissue sections. As the important role of non-coding RNAs in many biological processes becomes more and more appreciated, we foresee that the same protocol can likely be used to study other non-coding RNAs in the context of their specific chromatin.

Studying cells and tissues by means of fluorescent in situ hybridization (FISH) has, since its introduction in the late 1970s1, allowed researchers to study genes, chromatin organization and gene expression at the subcellular level. DNA FISH is frequently used in cytogenetics, karyotyping2, cancer diagnostics3 and pre-implantation genetic screening4, and has an important role in molecular research5-6 since it allows the detection of nucleic acids in their native environment. Single cell expression analysis by RNA FISH can detect native primary transcripts and noncoding RNAs being transcribed from chromosomes, and offers advantages to other techniques which assess gene expression at a population level, including for example quantitative RT-PCR, genome wide expression analysis or Northern blotting. By visualizing RNA transcripts originating directly from their sites of origin, it has for example been noticed that gene expression is stochastic7, and can sometimes be allele specific8. Improvements in the technique have even allowed the detection and quantification of single mRNA molecules within cells9-11.
The basic principle of FISH consists of hybridization of nucleic acids within the cell to a nucleic acid probe by means of highly specific Watson and Crick base-pairing. The probe can be either directly or indirectly detected, resulting in a signal which can be microscopically visualized. Initial attempts consisted of radioactively labeled probes, which had drawbacks based on safety issues, limited spatial resolution and the ability to detect only one target at a time12-14. The subsequent development of nonradioactive labels, including fluorochromes, haptens, and enzymes, has allowed the wide spread use of FISH as a routine molecular biology technique. The FISH probe can either be directly labeled with fluorochromes by chemical linking of fluorescent molecules to nucleic acid sequences15 and integration of fluorescently labeled nucleotides16-19, or the probe can be indirectly visualized after integration of haptens (including biotin and digoxigenin) and immunological detection of haptens by hapten specific antibodies conjugated to fluorescent reporter molecules20-21. The latter approach allows signal amplification by using several layers of fluorescently labeled antibodies which are used to enhance the original signal, and enables the detection of RNA species which are expressed at low levels. By combining direct and indirect labeling techniques and various haptens, several targets can be simultaneously visualized within the same cell.
One of the most important steps in FISH protocols is the hybridization of the probe to its target. Although in theory, a probe will specifically bind only to its target, in practice, this specificity is not always achieved, as probes may bind to homologous regions, and hybridization conditions will not always be ideal for a certain DNA region or RNA species. Post-hybridization washes are therefore of particular importance, as they can increase the stringency of the FISH procedure, and can prevent nonspecific binding of FISH probes, which would result in a high level of background noise. As RNA molecules are single stranded they can easily be hybridized to a FISH probe. In contrast, the double stranded DNA molecule first needs a denaturation step, after which a probe can hybridize. This is usually achieved by heating of the specimen, which results in denaturation of the DNA. However, under these harsh conditions, fragile, single stranded RNA molecules might be lost. Therefore, combined DNA-RNA FISH requires significant optimization of the conditions, and is more technically challenging compared to the separate detection of only RNA or DNA.
Here we present a detailed protocol of combined, simultaneous DNA-RNA FISH which has allowed us to study X chromosome inactivation (XCI) in differentiating female mouse embryonic stem cells22-24. XCI is a crucial epigenetic mechanism for female embryonic development25, and results in heterochromatinization and hence silencing of one of the two X chromosomes in female individuals26-27. Essential to this process is the noncoding RNA Xist28-30, which is regulated by the RNF1222-23 and REX124 proteins. Xist expression becomes upregulated on the future inactive X chromosome (Xi) during embryonic development or upon ES cell differentiation in vitro, and can spread along the X chromosome and thereby attract chromatin remodeling enzymes which result in the transcriptional shutdown of the X chromosome31. This spreading of Xist RNA can be visualized by RNA FISH as a coating of the X chromosome, which is also referred to as an Xist cloud. Since female ES cells can lose one of their X chromosomes due to genomic instability, we and others have employed combined DNA-RNA FISH to study XCI, to make sure that only karyotypically stable cells are assessed in the analysis of this important process22,32-34. As with every molecular biology technique, several different excellent protocols have been published35-38. Here we present our method, starting from the induction of differentiation in mouse ES cells, fixation of cells, labeling of FISH probes by Nick-translation, pre-treatment of fixed cells to allow permeabilization and subsequent probe uptake, hybridization of the probe to the target, and finally detection of the probe by fluorescently labeled antibodies. The herein presented protocol allows the faithful detection of Xist RNA and the X chromosome within a period of two days, and the basics of this technique can likely be adapted to other systems and areas of research.

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">female mouse embryonic stem cells</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">will be made available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse embryonic fibroblasts</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>can be derived from E13.5 embryos, or via commercial sources e.g Millipore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell culture medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>DMEM, 15% foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 0.1 mM &#223;-mercaptoethanol, and 1000 U ml-1 LIF. Filter sterilize and store at 4&#186;C for maximal 2 weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">LIF</td>
			<td style="width:124px;">Chemicon</td>
			<td style="width:119px;">ESG1107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">DMEM</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">11960085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Fetal Calf Serum&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Penicillin&#8211;streptomycin (100&#215;)</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Non-essential aminoacids&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">&#223;-mercaptoethanol&#160;</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell differentiation medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>IMDM-Glutamax, 15% heat inactivated foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 37.8 &#956;l/l monothioglycerol and 50 &#956;g/ml ascorbic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">IMDM-Glutamax</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">31980-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Ascorbic acid (50 mg/ml)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">monothioglycerol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">T25 cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">690160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">6-well cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">cell culture grade PBS</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Trypsin-EDTA 0.25% (v/v) trypsin/0.2% (w/v) EDTA in PBS&#160;</td>
			<td style="width:124px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">falcon tubes</td>
			<td style="width:124px;">Falcon</td>
			<td align="right" style="width:119px;">352196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">24x24 mm coverslips</td>
			<td style="width:124px;">Menzle</td>
			<td align="right" style="width:119px;">780882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">gelatin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>prepare a 0.2% gelatin/cell culture PBS solution, and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">paraformaldehyde</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>prepare 4% PFA/PBS solutions, by dissolving PFA in PBS. Stir at 70 &#7484;C until completely dissolved and cool down to room temperature. Aliquots can be stored at -20&#7484;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">absolute 100%&#160; ethanol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">32221-2.5L</td>
			<td>use absolute 100% ethanol and RNAse free water to make 70% and 90% ethanol solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">RNAse free water</td>
			<td style="width:124px;">Baxter</td>
			<td>TKF7114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">sterile filter tips</td>
			<td style="width:124px;">Rainin Bio Clean</td>
			<td style="width:119px;">GP-L10F, GP-L200F, RT-1000F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">digoxigenin-labeling kit (DIG-Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745816910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">biotin-labeling kit (Biotin Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745824910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Sephadex G50</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2 ml syringe</td>
			<td style="width:124px;">BD Plastipak</td>
			<td align="right" style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">sterilized cotton</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf tubes</td>
			<td style="width:124px;">Eppendorf</td>
			<td align="right" style="width:119px;">30120086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">yeast tRNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15401-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Salmon Sperm DNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15632-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse cot-1 DNA&#160; 1 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf bench top microcentrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5417R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Eppendorf Refrigerated Centrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5810R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M NaAc, pH 5.6</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">50+ hybridization solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>50% formamide, 2x SSC, 50 mM phosphate buffer pH 7, 10% dextran sulfate pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">formamide</td>
			<td style="width:124px;">Acros organics</td>
			<td align="right" style="width:119px;">3272350000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">dextran sulfate</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">31403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">pepsin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6887</td>
			<td>prepare a 0.2% solution in RNAse free water, and add 84 &#181;l 37% HCL per 100 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">HCL, 37% solution&#160;</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">84422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">object glasses</td>
			<td style="width:124px;">Starfrost</td>
			<td style="width:119px;">8037/1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">20x SSC</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">AM9763</td>
			<td>dilute to 2x SSC in RNAse free water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">10 mM Phosphate buffer, pH 7</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>add 57.7 ml of 1M Na2HPO4 and 42.3 ml 1M NaH2PO4; use DEPC treated water and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">denaturation buffer</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>70% Formamide/2xSSC/10mM phosphate buffer pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tween-20</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M Tris pH 7.5 solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tris-Saline-Tween washing solution (TST)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>100 ml 10x Saline, 50 ml 2M Tris, 500 &#181;l Tween-20, add RNAse free water till 1 liter&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">10x Saline solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>dissolve 85 g NaCl in 1 liter RNAse free H2O, and autoclave</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline-BSA (TSBSA)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>500 &#956;l 10x Saline, 250 &#956;l 2 M Tris, 1 ml 100x BSA, 3,25 ml RNAse free H2O</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">BSA, purified, 100x&#160;</td>
			<td style="width:124px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">sheep-anti-dig antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:500 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit-anti-sheep antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-rabbit antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse-anti-biotin antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:200 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">donkey-anti-mouse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-horse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline washing solution (TS)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>10 ml 10x Saline, 5 ml 2 M Tris, 85 ml RNAse free H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting medium with DAPI</td>
			<td style="width:124px;">Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nail-varnish</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">fluorescent miscroscope</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the above mentioned protocol for combined DNA-RNA FISH, we have been able to visualize X chromosome inactivation in differentiating female embryonic stem cells. Figure 1 shows a representative example of an DNA-RNA FISH experiment, where we detected both Xist (which is visible as an so-called Xist RNA cloud on the inactive X chromosome and a basal transcription pinpoint on the active X chromosome), and a region of the X chromosome, which is visible as a pinpoint signal. Note that one of the pinpoint signals is located within the Xist cloud, thereby showing that the Xist cloud is indeed localizing along, and coating the X chromosome. In more than 99% of cells, we detect using our protocol a correct DNA signal (data not shown). Compared to RNA-FISH, Xist clouds visualized using the combined DNA-RNA FISH procedure look sometimes larger, as the denatured DNA seems to occupy a larger area. We have obtained similar results using human ES cells, human lymphocytes and various fibroblast cell lines of various species, and the same protocol has been applied to study gene expression of various X-linked loci, including Rnf12. In addition, when this protocol was applied to undifferentiated female ES cells, basal Xist expression from both X chromosomes could be detected, which shows that using this protocol also genes expressed at low levels can be visualized (data not shown).
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51628/51628fig1highres.jpg" width="600" /> 
Figure 1. Combined DNA-RNA FISH in differentiated female ES cells. Representative results obtained after the herein described protocol for combined DNA-RNA FISH. The X chromosome (X chr.) is detected using a digoxigenin-labeled BAC probe, which is visualized using antibodies conjugated to FITC (green signal). In almost every cell, two X chromosomes are detected. Xist RNA is detected using a biotin labeled cDNA probe, which is visualized using antibodies conjugated to rhodamine (red signal). Both large Xist accumulations on the inactive X chromosome (Xist clouds) and basal Xist expression from the active X chromosome (Xist pinpoints) are detected (note: upon differentiation this basal Xist expression is ceased on the future active X chromosome, and therefore not detected in every nucleus). Nuclei are counterstained with DAPI (blue). Scale bar represents 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/51628/51628fig1highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells at JoVE.com

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.

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 ...

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27.5K Views.  Erasmus MC - University Medical Center. 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.

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...