We thank Zhaohua Irene Tang in the W. M. Keck Science Department for the use of her epifluorescence microscope and the Werren lab for donating Nasonia for dissections. This work was supported in part by an NIH-NRSA fellowship (5F32GM105317-02) to AML.

1. Tissue Dissection and Fixation (60 min)
<ol>
	<li>For Drosophila brains, place 3rd instar larvae in a drop of 1x PBS (phosphate buffered saline). Choose large 3rd instar larvae that are actively crawling from vials or bottles that are not overcrowded.
	<ol>
		<li>Use one ultrafine tweezer pair to grab hold of the mouth hooks and another tweezer pair to grab 2/3 down the length of the body (Figure 1A, B). Pull gently on the mouth hooks to expose the brain, ventral ganglia, salivary glands and part of the larval digestive tract. Use the tweezers to separate the brain and ventral ganglia (Figure 1A, B) from the other tissues and place in a droplet of 1x PBT (phosphate buffered saline with Tween) on a plastic Petri plate.</li>
	</ol>
	</li>
	<li>For Nasonia testis dissections, choose male 3-day-old pupae (yellow bodies with red eyes). Male Nasonia have small wing pad lengths relative to females during the pupal stage (Figure 1C).
	<ol>
		<li>Hold the pupa at the top of the abdomen near the thoracic region with one tweezer pair, and using the other tweezer pair, grab the very distal tip of the abdomen and pull out the tear-drop-shaped testes (they will be surrounded by fat body that can be gently shaken away; Figure 1D). Detach any exterior body parts from the testes, which will prevent proper squashing, and place in a droplet of 1x PBT on a plastic Petri plate.</li>
	</ol>
	</li>
	<li>For each slide, dissect a total of four or five tissue samples (i.e., Drosophila larval brains or Nasonia testes). 
	NOTE: More than five samples will lead to overcrowded nuclei and chromosomes.</li>
	<li>Optionally, to achieve some separation of sister chromatids in euchromatic regions of mitotic chromosomes, treat the tissue with a hypotonic solution: transfer brains from 1x PBT to a drop of 0.5% sodium citrate for 5-10 (no more than 10) min. 
	NOTE: Colchicine (a mitotic inhibitor) can be helpful for increasing the number of mitotic figures at metaphase11. However, its use is unnecessary if very large healthy larvae are used, and even undesirable because it can negatively affect the chromosome resolution, spreading and morphology.</li>
	<li>Place a droplet (~20 &#181;l) of fixative solution (2.5% paraformaldehyde in 45% acetic acid) onto the surface of a clean Sigmacote-treated cover slip. 
	NOTE: Make fixative solution fresh for each day of use. Fixative solutions ranging from 1.8-3.7% paraformaldehyde in 45% acetic acid yield the best results for FISH. Using tissues other than brains or testes, or adapting this protocol for immuno-FISH may require experimenting with different fixatives (for a list of different fixatives, see12).</li>
	<li>Carefully transfer each tissue sample from the dissecting buffer (1x PBT) into the fixative droplet with ultrafine tweezers, minimizing the transfer of dissecting buffer into the fixative solution. Position the tissue samples so that they are evenly spaced from one another within the fixative droplet. Incubate the tissues in fixative for 4 min at room temperature.</li>
	<li>Carefully place a poly-lysine-coated slide face down onto the tissue and cover slip. Do not press down at this point but instead allow the two to contact lightly so that the cover slip sticks onto the underside of the slide. Invert the slide so that the cover slip is on top.</li>
	<li>Sandwich the slide/tissue/cover slip inside a folded piece of filter paper. On a stable surface, using the thumb, press very firmly straight down onto the position directly above the cover slip. Be careful to avoid lateral sliding of the cover slip (this will cause smearing of the tissue).</li>
	<li>Submerge the slide/tissue/cover slip into liquid nitrogen (see apparatus in Figure 1E, F), and let stand until the Nitrogen stops boiling (longer is fine). Remove the slide and immediately snap off the cover slip with a fresh razor blade by flicking a corner of the cover slip in an upward direction (avoid scratching the fixed tissue with the razor blade).
	<ol>
		<li>Pre-cooling the slide/tissue/cover slip on a block of dry ice, cover slip up, for 1-2 min prior to submerging in liquid nitrogen will help prevent the slides from cracking.</li>
	</ol>
	</li>
	<li>Immediately place the slide with tissue into a Coplin jar filled with 100% ethanol at room temperature and let stand for at least 5 min (this time can be longer if needed). 
	NOTE: Cold 100% ethanol could also be used.</li>
	<li>Remove the slide with tissue, wick away excess ethanol with a Kimwipe (without touching the fixed tissue), and leave the slide to air dry for 1 hr.
	<ol>
		<li>Proceed directly to step 2 or keep slides dry in low humidity air or in a desiccation chamber for weeks to even months before performing the hybridization.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52288/52288fig1highres.jpg" /> 
Figure 1: (A) A 3rd instar Drosophila larva (right), with positions indicated for where to grab the mouth hooks (denoted with *) and 2/3 of the way down the larvae to dissect brains; (left) a schematic of a larva a the same developmental stage, depicting the relative position of the brain within the larval head. (B) The brain and ventral ganglia dissected from a 3rd instar Drosophila larva (right) and a schematic of this tissue (left). (C) 3-day-old Nasonia pupa at the yellow body-red eye stage. (D) A testis pair dissected from a 3 day old Nasonia male pupa (right) with positions indicating where to grab the pupa at the posterior of the abdomen (denoted with *) and midway on the body; (left) schematic depicting male and female wasp pupae; male pupae can be distinguished by wings that do not extend past the saggital profile (black arrow), in contrast to females, which have wings that extend past the profile; the relative position of the testis pair is shown in the male pupa. (E) The apparatus&#8212;a string of paperclips&#8212;and (F) method used to immerse slides in a vessel of liquid nitrogen. Multiple paperclip strings can be used for simultaneous immersion of multiple slides.
2. In situ Hybridization (30 min on day 1; 1 hr&#8212;2.5 hr for long probes&#8212;on day 2)
<ol>
	<li>Add 1 &#181;l (100 ng) of each probe to 20 &#181;l of 1.1x hybridization buffer. Pipet probe/hybridization buffer onto the surface of the fixed tissue (avoid touching the tissue).</li>
	<li>Carefully place a cover slip directly onto the probe/hybridization buffer, making sure that the cover slip is centered directly over the tissue. The buffer should migrate to the outer edge of the cover slip, leaving no air bubbles. 
	NOTE: Small bubbles that do not contact tissue do not pose any problems to the procedure. Remove large bubbles by carefully lifting a corner of the cover slip and carefully dropping it back onto the slide.</li>
	<li>Place the slide/tissue/cover slip onto the surface of a block pre-heated to 95 &#176;C (cover slip up). Cover with a large piece of aluminum foil to prevent light exposure. Let the slide incubate at 95 &#176;C for 5 min. 
	NOTE: A typical heat block with holes for tubes can be flipped over to provide a flat surface on which to place the slide/tissue/cover slip.</li>
	<li>Remove the slide, letting it cool slightly until it is warm to the touch. Carefully wrap a piece of stretched Parafilm around the cover slip to seal the liquid beneath it.</li>
	<li>Place the sealed slide inside a humidity chamber and place the chamber into an incubator preheated to 30 &#176;C. Incubate at 30 &#176;C for 4 hr to overnight.
	<ol>
		<li>Create a humidity chamber from an empty tip box or lidded Tupperware container with dampened Kimwipes or paper towels placed at the bottom. 
		NOTE: Single stranded DNA oligo probes have been designed to be 28-33 bases to achieve a theoretical melting temperature (Tm) of 45-47 &#176;C. These length and Tm ranges reflect the fact that many repetitive sequences that we have studied are AT-rich and therefore have very low GC content. Longer probes will likely have higher Tm values; this may result in higher background hybridization at the standard hybridization temperature of 30 &#176;C. Thus, some troubleshooting with hybridization temperatures may be required to achieve the best results. To find the best hybridization temperature, increase (or decrease) the temperature by 5 &#176;C, incrementally.</li>
	</ol>
	</li>
	<li>Carefully remove the Parafilm from the slide and then carefully remove the cover slip by slowly lifting one corner. Wash the slide three times for 15 min each wash in 0.1x SSC buffer. Cover the Coplin jar with aluminum foil during the washes to minimize light exposure.</li>
	<li>If not using a long biotinylated probe, proceed to step 2.8.
	<ol>
		<li>If using a long biotinylated probe, dry the area around the tissue with a Kimwipe, being careful not to touch the tissue itself. Place 100 &#181;l of blocking solution over the tissue and cover gently with a coverslip, taking care to avoid trapping bubbles. Wrap the slide over the coverslip with Parafilm and place at 37 &#176;C for 30 min.</li>
		<li>Carefully remove the coverslip and blot around the tissue with a Kimwipe. Pipet 100 &#181;l of rhodamine-avidin diluted 1:1,000 in SBT over the tissue and cover gently with a coverslip, taking care to avoid trapping bubbles. Wrap the slide over the coverslip with Parafilm and place at 37 &#176;C for 30 min.</li>
		<li>Carefully remove the coverslip and wash the slide 3 times for 5 min each in 4x SSCT and then 3 times for 5 min each in 0.1x SSC. 
		NOTE: Slides can be washed for longer periods of time.</li>
	</ol>
	</li>
	<li>Remove the slide and blot around the tissue with a dry Kimwipe to remove excess buffer (avoid touching the tissue). Place the slide tissue side up in a dark place for 10-15 min or until the moisture completely dissipates.</li>
	<li>Pipet 11 &#181;l of Vectashield mounting medium (with 4&#8217;,6-diamidino-2-phenylindole&#8212;DAPI) onto the tissue. Carefully place a clean cover slip (not treated with Sigmacote) directly over the center of the mounting medium and tissue. The mounting medium should migrate slowly outward toward the edges of the cover slip. 
	NOTE: If the mounting medium fails to reach the edge of the cover slip on all sides, then an additional 1-2 &#181;l of mounting medium can be applied to one position at the edge of the cover slip to fill in the needed volume. In this case, be sure to wipe away any excess medium from the slide surface before sealing.</li>
	<li>Seal the edges of the cover slip with nail polish. Avoid painting the nail polish over the tissue sample.</li>
	<li>Place the slide upright in a dark place and let the nail polish dry until completely hard (usually 30 min or more). At this point, image the tissue or store at -20 &#176;C for up to 1 week for later imaging.</li>
</ol>
Buffer/Solution Recipes
10x PBS
<ul>
	<li>80 g NaCl</li>
	<li>2.0 g KCl</li>
	<li>14.4 g Na2HPO4</li>
	<li>2.4 g KH2PO4</li>
	<li>pH to 7.4, H2O to 1 L</li>
</ul>
1x PBT
<ul>
	<li>5 ml 10x PBS</li>
	<li>45 ml H2O</li>
	<li>0.1% Tween 20</li>
</ul>
20x SSC
<ul>
	<li>175.3 g NaCl</li>
	<li>88.2 g Na Citrate</li>
	<li>in 800 ml H2O</li>
	<li>pH to 7, H2O to 1 L</li>
</ul>
4x SSCT
<ul>
	<li>200 ml 20x SSC</li>
	<li>799 ml H2O</li>
	<li>0.1% Tween 20</li>
</ul>
0.1x SSC
<ul>
	<li>5 ml 20x SSC</li>
	<li>995 ml H2O</li>
</ul>
Hybridization mix (20 &#181;l; modified from 11)
<ul>
	<li>10 &#181;l formamide&#160;</li>
	<li>4 &#181;l 50% dextran sulfate</li>
	<li>2 &#181;l 20x SSC</li>
	<li>4 &#181;l H2O</li>
</ul>
SBT8 (10 ml)
<ul>
	<li>2 ml 20x SSC</li>
	<li>0.01 g Bovine Serum Albumin (BSA)</li>
	<li>10 &#181;l Tween 20</li>
	<li>7.9 ml H2O</li>
</ul>
Blocking solution8 (10 ml)
<ul>
	<li>0.3 g BSA</li>
	<li>10 &#181;l Tween 20</li>
	<li>2 ml 20x SSC</li>
	<li>8 ml H2O</li>
</ul>
Fixative solution with paraformaldehyde (1 ml)
<ul>
	<li>393.75 &#181;l H2O (add water first)</li>
	<li>450 &#181;l glacial acetic acid</li>
	<li>156.25 &#181;l 16% paraformaldehyde</li>
</ul>

<ol>
	<li>Blattes, R., Kas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+(FISH)+on+diploid+nuclei+and+mitotic+chromosomes+from+Drosophila+melanogaster+larval+tissues.">Fluorescent in situ hybridization (FISH) on diploid nuclei and mitotic chromosomes from Drosophila melanogaster larval tissues.</a> Cold Spring Harbor Protocols. 2009 (9), (2009).</li><li>Dimitri, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+with+transposable+element+probes+to+mitotic+chromosomal+heterochromatin+of+Drosophila.">Fluorescent in situ hybridization with transposable element probes to mitotic chromosomal heterochromatin of Drosophila.</a> Methods in Molecular Biology. 260, 29-39 (2004).</li><li>Pardue, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+to+polytene+chromosomes+in+Drosophila+using+digoxigenin-labeled+probes.">In situ hybridization to polytene chromosomes in Drosophila using digoxigenin-labeled probes.</a> Cold Spring Harbor Protocols. 2011 (8), 1003-1006 (2011).</li><li>Hoskins, R. 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=Heterochromatic+sequences+in+a+Drosophila+whole-genome+shotgun+assembly.">Heterochromatic sequences in a Drosophila whole-genome shotgun assembly.</a> Genome Biology. 3 (12), (2002).</li><li>Hoskins, R. 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=Sequence+finishing+and+mapping+of+Drosophila+melanogaster+heterochromatin.">Sequence finishing and mapping of Drosophila melanogaster heterochromatin.</a> Science. 316 (58331), 1625-1628 (2007).</li><li>Treangen, T. J., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repetitive+DNA+and+next-generation+sequencing:+computational+challenges+and+solutions.">Repetitive DNA and next-generation sequencing: computational challenges and solutions.</a> Nature Reviews Genetics. 13 (1), 36-46 (2012).</li><li>He, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+pericentric+heterochromatin+by+comparative+genomic+hybridization+analysis+and+chromosome+deletions+in+Drosophila+melanogaster.">Mapping the pericentric heterochromatin by comparative genomic hybridization analysis and chromosome deletions in Drosophila melanogaster.</a> Genome Research. 22 (12), 2507-2519 (2012).</li><li>Pimpinelli, S., Bonaccorsi, S., Fanti, L., Gatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+in+situ+hybridization+(FISH)+of+mitotic+chromosomes+from+Drosophila+larval+brain.">Fluorescent in situ hybridization (FISH) of mitotic chromosomes from Drosophila larval brain.</a> Cold Spring Harbor Protocols. 2010 (3), (2010).</li><li>Larracuente, A. M., Noor, M. A., Clark, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+Y-linked+genes+to+the+dot+chromosome+in+Drosophila+pseudoobscura.">Translocation of Y-linked genes to the dot chromosome in Drosophila pseudoobscura.</a> Molecular Biology and Evolution. 27 (7), 1612-1620 (2010).</li><li>Ferree, P. M., Barbash, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+heterochromatin+prevents+mitotic+chromosome+segregation+to+cause+hybrid+lethality+in+Drosophila.">Species-specific heterochromatin prevents mitotic chromosome segregation to cause hybrid lethality in Drosophila.</a> PLoS Biology. 7 (10), e1000234 (2009).</li><li>Williams, B. C., Karr, T. L., Montgomery, J. M., Goldberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+l(1)zw10+gene+product,+required+for+accurate+mitotic+chromosome+segregation,+is+redistributed+at+anaphase+onset.">The Drosophila l(1)zw10 gene product, required for accurate mitotic chromosome segregation, is redistributed at anaphase onset.</a> The Journal of Cell Biology. 118 (4), 759-773 (1992).</li><li>Gatti, M., Bonaccorsi, S., Pimpinelli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+Drosophila+Mitotic+Chromosomes.">Looking at Drosophila Mitotic Chromosomes.</a> Methods in Cell Biology. 44, 371-391 (1994).</li><li>Werren, J. H., Stouthamer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PSR+(paternal+sex+ratio)+chromosomes:+the+ultimate+selfish+genetic+elements.">PSR (paternal sex ratio) chromosomes: the ultimate selfish genetic elements.</a> Genetica. 117 (1), 85-101 (2003).</li></ol>

The authors declare that they have no competing financial or any other conflict of interest.

DNA ISH is frequently used to map specific sequences to chromosomes. We have described a simple method for DNA ISH optimized for high copy number, heterochromatic sequences. Rather than using washes in a formamide solution, which is a requirement in other existing DNA ISH protocols, we place tissue-mounted slides directly on a pre-heated block to denature DNA. This method circumvents the use of large amounts of formamide. One critical step for producing crisp hybridization signals is to use freshly made fixation solution...

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. Probes to single copy regions within euchromatin can be generated through a handful of approaches, including nick-translation or end-labeling of long DNA products1,2 and the incorporation of deoxygenin (DIG)-attached nucleotides and their recognition through a wide variety of group-conjugated antibodies1-3. Visualization of euchromatic sequences in few or single copy number requires the use of either single, large probes with high specific activity or a cocktail of multiple, smaller probes that collectively enhance signal.
In contrast, highly repetitive sequences found in heterochromatin, such as satellite DNAs, are easier targets for DNA ISH because they normally exist as tens to thousands of repeats clustered in single chromosome regions known as blocks. Transposable elements also can be found at high copy numbers at distinct chromosomal loci2. In these cases, single probes with low specific activity can effectively label heterochromatic sequences due to their hybridization at multiple sites. Probes to repetitive sequences can be synthesized commercially as short oligonucleotides (30-50 bp) and chemically conjugated with any of multiple different fluorescent groups. Mapping repetitive sequences within heterochromatin by using genome-sequencing technologies is difficult due to challenges encountered in building scaffolds within highly repetitive satellite blocks4-6,7. Currently, ISH stands as the most effective way of mapping these sequences at the sub-chromosome level. This strategy is important for mapping large numbers of repetitive sequences that are being uncovered by ongoing genome and transcriptome sequencing studies.
The efficiency and ease of mapping repetitive sequences on slide-mounted chromosomes would be greatly enhanced by a simplified protocol for DNA ISH. For example, existing protocols for DNA ISH involve multiple washes of hybridized tissues in formamide solution2,8, thus adding substantially to the required time for mapping sequences and also producing large amounts of chemical waste for this costly reagent. Here we describe a revised DNA ISH method that circumvents the need for formamide washes and utilizes basic laboratory equipment and reagents. This method was originally designed for the rapid mapping of highly repetitive DNA sequences in heterochromatic regions of Drosophila larval neuroblasts by using commercially synthesized oligos that are conjugated with fluorescence dyes. However, this method also works for mapping repetitive sequences by using larger probes synthesized through other means9,10 and across multiple different tissue and chromosome types. Additionally, this method can be used to map euchromatic sequences by using longer or multiple, short probes within the euchromatic sequence of interest.

<table border="0" cellpadding="0" cellspacing="0" width="542">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:285px;"></td>
			<td style="width:257px;">Company</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Materials&#160;</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Poly-L-lysine coated slides (regular slides also can be used)</td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ultrafine tweezers (5 gauge)</td>
			<td>Dumont</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">22mmx22mm cover slips, Sigmacote-treated by immersion for 15 seconds, blotting dry, and wiping away all traces of Sigmacote so that cover slip is clear</td>
			<td>Fisher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sigmacote</td>
			<td>Sigma</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Filter paper (75-150mm)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraffin wax paper</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat block with thermometer</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dry incubator</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Razor blades</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Humidity chamber (empty pipette tip box or Tupperware, lined with moistened paper towels or Kimwipes)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coplin jars (with slide grooves)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Aluminum foil</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pasteur pipettes</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1.5 mL microfuge tubes</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nail polish (clear or colored)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">2-20 &#956;L micropipette and plastic tips</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">20-25 standard metal paperclips linked to form a chain</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td>Company/Notes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Reagents</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">16% EM grade paraformaldehyde</td>
			<td>Electron Microscopy Reagents</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acetic acid</td>
			<td>Sigma</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Liquid nitrogen</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">100% Ethanol, chemical grade</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Commercially synthesized, fluorescently labeled oligos</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Long biotinylated probe (e.g. nick translated and biotinylated with BioNick from Invitrogen)</td>
			<td>Invitrogen; Alternative steps 2.7.1-2.7.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rhodamine-Avidin (for detection of long biotinylated probe)</td>
			<td>Roche; Alternative steps 2.7.1-2.7.3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hybridization buffer</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">4X SSCT (Saline-sodium citrate + Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">0.1X SSC (Saline-sodium citrate)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Blocking solution</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">SBT (SSC, Bovine serum albumin, Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBT (Phosphate-buffered saline + Tween)</td>
			<td>Recipe below</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBS (Phosphate-buffered saline)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td colspan="2" height="17" style="height:17px;">Hypotonic solution (0.5% sodium citrate in H2O)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formamide</td>
			<td>Sigma Aldrich</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vectashield mounting medium with DAPI</td>
			<td>Vector laboratories</td>
		</tr>
	</tbody>
</table>

simple method for fluorescence dna in situ hybridization to squashed chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

To demonstrate this method, we hybridized a set of small commercially synthesized oligos that were chemically modified with fluorescent conjugates (Figure 2) and a longer biotinylated probe (made through nick translation of a PCR product; Figure 2B), to chromosomes from several different tissue types (see Table 1). The target sequences included satellite repeats located in pericentromeric (heterochromatic) regions of mitotic chromosomes from D. melanogaster larv...

Watch this Scientific Journal Video about Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes at JoVE.com

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

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18.0K Views.  University of Rochester. Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Video: Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

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 ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

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 ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

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

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

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

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

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

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...