Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C1277).

1. Labeling Probes with Digoxigenin-dUTP by PCR
<ol>
	<li>Perform PCR labeling with 10x dNTP mix containing digoxigenin-dUTP as previously described13.</li>
	<li>Purify PCR product with spin-column purification according to manufacturer&#39;s instruction.
	<ol>
		<li>If the probe is shorter than 200 bp, remove free digoxigenin-dUTP with spin-column chromatography from the reaction mixture rather than spin-column purification.</li>
	</ol>
	</li>
</ol>
2. Preparing Polylysine Coated Slides
Note: The entire procedure takes about 2 hr. Most of the steps are done at room temperature except for the drying step.
<ol>
	<li>Cleaning the slides
	<ol>
		<li>Place the slides in a plastic container and rinse the slides briefly with distilled water (DW). Remove the water and fill the container with DW containing 1% glass cleaner.</li>
		<li>Agitate the slides for 15 min at 50 rpm at room temperature (RT). Wash the slides with DW 3 times for 5 min each at RT.</li>
		<li>Wash the slides with 70% ethanol for 15 min with agitation. Discard 70% ethanol and place the slides on a 65 &#176;C dry block and air-dry for 15 min.</li>
	</ol>
	</li>
	<li>Polylysine coating of multi-well glass slides 
	Note: Polylysine coating of slide glass is an important step, since it provides the sample adhesion throughout the staining procedure. Poorly coated slide will result in the loss of sample.
	<ol>
		<li>Dilute the polylysine stock solution to 0.01% (w/v) in distilled water. Add 20 &#181;l of the diluted 0.01% (w/v) polylysine to the wells of a clean glass slide.</li>
		<li>Incubate the glass slide for 5 min at RT.</li>
		<li>Place the slides on a 65 &#176;C dry block and air-dry for 1 hr.</li>
		<li>Store the polylysine slides in the dust free box.</li>
	</ol>
	</li>
</ol>
3. Fixation of Worms on the Slide Glass (Figure 1)
<ol>
	<li>Preparing embryos for FISH 
	Note: Harvest worms before all the bacterial food is consumed by watching the growth media under microscope. Starvation reduces egg production of adult worms and increases egg hatching. Detailed methods are described in 14,15.
	<ol>
		<li>Grow the worms in 50 mm Petri-dish according to standard methods14.</li>
		<li>After all the bacterial food is consumed, cut the agar media in quarter with spatula. Sterilize the spatula before cutting to prevent contamination.</li>
		<li>Put all the piece of agar on the 100 mm nematode growth media (NGM) plate. Turn the agar piece upside down for the worms to reach the fresh bacterial food.</li>
		<li>After 48 to 72 hr, collect the worms with M9 buffer. Add 3 - 5 ml of M9 buffer on the NGM plate. Pipette M9 buffer on the surface of NGM to wash the worms.</li>
		<li>Collect the liquid with worms and add to a 15 ml tube. 
		Note: If there is agar debris after harvest, centrifuge the worms in a 30% sucrose solution. While debris are pelleted, worms float on the surface.</li>
		<li>Add M9 buffer to make up the volume to 15 ml. Pellet the worms by centrifugation at 300 x g for 3 min and remove most of the M9 buffer. Repeat this step 2 more times. 
		Note: If the worms are still floating after centrifugation, set the brake level of the centrifuge to value = 1.</li>
		<li>Aspirate M9 buffer. Add bleaching solution to the worms. Per 0.5 ml of worms, add 6.5 ml of DW, 2 ml of hypochlorite, 1 ml of 5 M KOH.</li>
		<li>Incubate the worms with rocking at RT for 3 min at 50 rpm. Vortex worms for 15 seconds to mechanically shear the worms and expose the eggs.</li>
		<li>Observe the tube under a dissection microscope during bleaching. Make sure that worms are cut in half and eggs are released. When the most of the adult body is dissolved, add M9 buffer to make up the volume to 15 ml.</li>
		<li>Centrifuge at 300 x g for 3 min. Aspirate most of the M9 and add fresh M9 buffer to make up the volume to 15 ml. Repeat wash step 3 times. 
		Note: Avoid using excessive amount of worms, as they hinder the bleaching process. Keep the overall reaction time less than 8 min until the wash. Over-bleached eggs produce strong autofluorescence.</li>
		<li>Add phosphate buffered saline with polysorbate-20 (PBST) up to 200 &#181;l and 200 &#181;l of 4% paraformaldehyde (PFA) to make 2% PFA. 
		Caution: Since PFA is carcinogenic, wear protective clothing, gloves and eye shield before using PFA.</li>
		<li>Add 40 &#181;l of the eggs in 2% PFA onto the well of polylysine coated slide.</li>
		<li>Place the slides in a humid chamber and incubate for 15 min at RT. Close the humid chamber right after the slides are placed inside. 
		Note: The eggs settle to the bottom of slide while being fixed.</li>
	</ol>
	</li>
	<li>Preparing dissected gonads for FISH
	<ol>
		<li>Harvest adult worms grown on 50 mm NGM plate by pipetting 1 ml of M9 buffer. Harvest worms before bacterial food is depleted.</li>
		<li>Wash the worms from any bacteria with M9 buffer, 2 times. 
		Note: Residual bacteria may interfere with the dissected gonads from sticking to polylysine coated slides.</li>
		<li>Pellet the worms by centrifugation at 300 x g. Remove M9 and transfer the worms to the empty NGM plate by micropipette.</li>
		<li>Add 30 &#181;l of M9 buffer containing 2 mM levamisole on a well of polylysine treated slide.</li>
		<li>Add 500 &#181;l of M9 buffer to a 1 ml tube. Use this buffer to transfer the worms by mouth pipette.</li>
		<li>Fill the tip of the mouth pipette with M9 buffer by placing a capillary in the 1 ml tube containing the M9 buffer.</li>
		<li>Under dissecting microscope, put the tip of mouth pipette just in front of the head of adult worm and drag mouth pipette so that the head of worms enters the mouth pipette. Once the head of worms enters the tip, the entire body of worm will be drawn into the tip.</li>
		<li>Transfer the worms to the polylysine coated slide using mouth pipette.</li>
		<li>Using a razor, cut off the head or the tip of the tail of worms on the slide. When the worm is cut, the gonads will pop out. Gonads will stick to the slides.
		<ol>
			<li>Prepare at least 30 worms in one well. More wells can be used for another 30 worms.</li>
		</ol>
		</li>
		<li>Put the slide in the humid chamber and aspirate off the M9 buffer with mouth pipette.</li>
		<li>Fix the sample by adding 20 &#181;l of 2% PFA at RT for 15 min in the humid chamber.</li>
	</ol>
	</li>
</ol>
4. Fixation and Permeabilization
<ol>
	<li>Place an aluminum block on dry ice and store it in a deep freezer (-80 &#176;C). Store methanol and acetone in -20 &#176;C.</li>
	<li>After PFA fixation step 3.1.15 or 3.2.11, remove fixative using micropipette leaving ~5 &#181;l of the fixative.</li>
	<li>Put another polylysine coated slide on the sample slide. Remove the fixative with paper towel if the solution is excessive. Do not move the slides once they are stuck together.</li>
	<li>Freeze the slides on the aluminum block for at least 15 min. 
	Note: The samples can be stored for at least 2 - 3 days.</li>
	<li>While the slides are being frozen, put the jars containing cold methanol and acetone on ice.</li>
	<li>Take the slides out and twist them to freeze-crack the sample. Discard the upper slide. Immediately soak the slide into the ice-cold methanol for 5 min.</li>
	<li>Transfer the slides to ice-cold acetone for 5 min.</li>
	<li>Wash the slides 3 times with PBST for 5 min to remove residual fixative. Proceed to the next step or store the slides in 100% ethanol at 4 &#176;C. 
	Note: The samples can be stored for at least 2 - 3 days.</li>
</ol>
5. Hybridization of Fixed Cells
<ol>
	<li>Add 20 &#181;l of RNase solution (PBST containing 10 &#181;g/ml RNase A). Incubate the slide in the humid chamber at 37 &#176;C for 1 hr.</li>
	<li>Wash the slide twice in 2x saline and sodium citrate with polysorbate-20 (2x SSCT) for 15 min each.</li>
	<li>Add 20 &#181;l of hybridization solution and put the humid chamber in the 37 &#176;C incubator. After 1 hr, remove the hybridization solution by pipetting.</li>
	<li>Before removing hybridization solution, prepare the probe. If the probe is double stranded DNA, denature the probes by heating at 95 &#176;C for 5 min on a dry block. After heating, cool the probe on ice briefly.</li>
	<li>Add 10 &#181;l of hybridization solution containing probes to the sample. For PNA probe, use concentration at a ratio of 1: 2,000 and for dig-labeled probe, use concentration at a ratio of 1:200. Cover the sample with cover glass.</li>
	<li>Put a paper towel soaked with water on the heat block (80 &#176;C). Put a plastic box cover on the heat block to preserve the humidity and temperature.</li>
	<li>After the temperature of the heat block has stabilized (to 80 &#176;C), place the sample slide on the heated paper towel and cover the samples with the plastic box cover. Denature the sample for 3 min.</li>
	<li>Incubate the slides in a humid chamber overnight at 37 &#176;C.</li>
</ol>
6. Washes and Immunofluorescence
<ol>
	<li>Warm up the hybridization wash solution (2x SSC, 50% formamide) to 37 &#176;C.</li>
	<li>Wash the sample in the PBST twice at RT for 5 min. Remove the cover glass.</li>
	<li>Wash the sample in hybridization wash solution at 37 &#176;C for 30 min.</li>
	<li>Wash the sample slide in PBST 3 times at RT. Note: Perform all the subsequent steps in humid chamber at RT.</li>
	<li>Add 20 &#181;l of blocking solution and incubate for 1 hr at RT in the humid chamber.</li>
	<li>Remove blocking solution and add FITC conjugated anti-digoxigenin antibody solution (1:200) for 3 hr at RT or overnight at 4 &#176;C.</li>
</ol>
7. Mounting and Observation
<ol>
	<li>Wash the sample slide with PBST 2 times for 15 min each.</li>
	<li>Add 10 &#181;l of mounting solution with DAPI. Put the cover glass and press gently. Remove any excess solution with a paper towel.</li>
	<li>To prevent evaporation of mounting solution, seal the edges of the cover glass with nail polish.</li>
	<li>Observe under confocal microscope. Exclude embryos with high background. Focus on a field with 4 - 20 nuclei.</li>
	<li>Take images according to manufacturer&#39;s instruction with 100X objective lens. 
	Note: Excite sample with 405 nm laser for DAPI, with 555 nm laser for cy3, with 488 nm laser for FITC.</li>
</ol>
8. Quantification of Telomere Signal
Note: Quantification was done as described previously16. All the images that are to be compared should be taken with same setting including exposure time and light source.
<ol>
	<li>Export the image in .tif format.</li>
	<li>Download and install the image analysis software.</li>
	<li>Execute the image analysis software and click agree button.</li>
	<li>Click open button. Open the images with telomere FISH by double-clicking the image file.</li>
	<li>Click [edit] - [select processing region], select region of interest by left-click and dragging. Exclude all the non-specific staining.</li>
	<li>Click [measure] - [spot optical densities], select the channel with telomere signal and enter the file name to save the results in .txt file. 
	Note: The column of results are in the following order: Fluorescence of spot, background intensity of spot and area of spot.</li>
	<li>Copy the values and subtract background intensity of spot from fluorescence of spot. The values can now be statistically analyzed.</li>
</ol>

<ol>
	<li>Reddel, R. R., Bryan, T. M., Murnane, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+cells+with+no+detectable+telomerase+activity.+A+review.">Immortalized cells with no detectable telomerase activity. A review.</a> Biochemistry-Moscow. 62, 1254-1262 (1997).</li><li>Seo, 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=Telomere+maintenance+through+recruitment+of+internal+genomic+regions.">Telomere maintenance through recruitment of internal genomic regions.</a> Nat Commun. 6, 8189 (2015).</li><li>Cesare, A. J., Reddel, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+lengthening+of+telomeres:+models,+mechanisms+and+implications.">Alternative lengthening of telomeres: models, mechanisms and implications.</a> Nat Rev Genet. 11, 319-330 (2010).</li><li>Meier, 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=trt-1+is+the+Caenorhabditis+elegans+catalytic+subunit+of+telomerase.">trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase.</a> Plos Genetics. 2, 187-197 (2006).</li><li>Cawthon, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+measurement+by+quantitative+PCR.">Telomere measurement by quantitative PCR.</a> Nucleic Acids Res. 30, 47 (2002).</li><li>Raices, M., Maruyama, H., Dillin, A., Karlseder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+of+longevity+and+telomere+length+in+C.+elegans.">Uncoupling of longevity and telomere length in C. elegans.</a> PLoS Genet. 1, 30 (2005).</li><li>Southern, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Specific+Sequences+among+DNA+Fragments+Separated+by+Gel-Electrophoresis.">Detection of Specific Sequences among DNA Fragments Separated by Gel-Electrophoresis.</a> Journal of Molecular Biology. 98, 503 (1975).</li><li>Lee, M., 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=Telomere+extension+by+telomerase+and+ALT+generates+variant+repeats+by+mechanistically+distinct+processes.">Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes.</a> Nucleic Acids Res. 42, 1733-1746 (2014).</li><li>Cheung, I., 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=Strain-specific+telomere+length+revealed+by+single+telomere+length+analysis+in+Caenorhabditis+elegans.">Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans.</a> Nucleic Acids Res. 32, 3383-3391 (2004).</li><li>Shtessel, L., 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=Caenorhabditis+elegans+POT-1+and+POT-2+repress+telomere+maintenance+pathways.">Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways.</a> G3. 3, 305-313 (2013).</li><li>Duerr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry.">Immunohistochemistry.</a> WormBook (The C. elegans Research Community). , (2006).</li><li>Phillips, C. M., McDonald, K. L., Dernburg, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+analysis+of+meiosis+in+Caenorhabditis+elegans.">Cytological analysis of meiosis in Caenorhabditis elegans.</a> Meiosis: Volume 2, Cytological Methods. , 171-195 (2009).</li><li>Emanuel, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+efficient+system+for+synthesis+of+non-radioactive+nucleic+acid+hybridization+probes+using+PCR.">Simple and efficient system for synthesis of non-radioactive nucleic acid hybridization probes using PCR.</a> Nucleic acids research. 19, 2790 (1991).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> J Vis Exp. , e4019 (2012).</li><li>Poon, S. S. S., Martens, U. M., Ward, R. K., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+length+measurements+using+digital+fluorescence+microscopy.">Telomere length measurements using digital fluorescence microscopy.</a> Cytometry. 36, 267-278 (1999).</li><li>Ferreira, H. C., Towbin, B. D., Jegou, T., Gasser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shelterin+protein+POT-1+anchors+Caenorhabditis+elegans+telomeres+through+SUN-1+at+the+nuclear+periphery.">The shelterin protein POT-1 anchors Caenorhabditis elegans telomeres through SUN-1 at the nuclear periphery.</a> J Cell Biol. 203, 727-735 (2013).</li><li>Lee, M. H., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+in+situ+hybridization+of+dissected+gonads.">RNA in situ hybridization of dissected gonads.</a> WormBook. , 1-7 (2006).</li><li>Tabara, H., Motohashi, T., Kohara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-well+version+of+in+situ+hybridization+on+whole+mount+embryos+of+Caenorhabditis+elegans.">A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans.</a> Nucleic Acids Res. 24, 2119-2124 (1996).</li><li>Poon, S. S., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescence+in+situ+hybridization+(Q-FISH).">Quantitative fluorescence in situ hybridization (Q-FISH).</a> Curr Protoc Cell Biol. 18, 14 (2001).</li></ol>

The authors have nothing to disclose.

The main advantage of our protocol is the simplicity of the procedure without noticeable damage to the morphology of cellular structure. Several steps were optimized for C. elegans FISH in this protocol. The critical steps for successful FISH include labeling of probes, fixation of embryos and penetration. Digoxigenin-dUTP labeling method provides an easy-to-use labeling method by PCR or nick-translation. To label long target sequence, nick-translation is preferred. In this case, the probes should be digested with appropriate restriction enzyme to facilitate the penetration of probes. Biotin-dUTP tag is not recommended because biotin-labeled probes produced excess amount of background signal from the cytoplasm. Although endogenous biotin blocking reagent is commercially available, it was not attempted.
This protocol uses isolated embryos to increase the density of cells for efficient quantification. Intestinal nuclei of C. elegans are large in size and are polyploid, which contribute excessive number and intensity of telomere spots compared to the rest of somatic nuclei. For this reason, whole worm is not suitable for quantification of telomeres in C. elegans. In contrast, embryos are appropriate for evaluation of telomere length as they provide homogeneous cells without effect of polyploidy.
This protocol uses 2% PFA fixative that worked fine for telomere FISH. Although glutaraldehyde is reported to result in a lower background signal and harder fixation in RNA in situ hybridization, glutaraldehyde increased autofluorescence significantly18. This excess background was not abolished after treatment of sodium borohydride, which reduces unreacted aldehyde group. For this reason, glutaraldehyde was not used. If the signal-to-noise ratio is low, time of prehybridization can be extended up to several hours to block non-specific binding sites. In addition, time of stringent washing can be increased to decrease background level.
Staining technique in C. elegans can be a challenge for proper permeabilization treatment. C. elegans contains thick cuticular exoskeleton which inhibits penetration of antibodies and probes. Traditional antibody staining method involves the treatment of collagenase, which requires much time and optimization process. The enzymatic penetration method also damages the morphology of the worms in exchange of penetration efficiency. Freeze-crack and methanol-acetone treatment was used to facilitate probe penetration. Freeze-crack is simple and rapid compared to chitinase or yatalase treatment19. Dehydration or rehydration of methanol series did not seem to affect the quality of FISH. These steps were simplified in this protocol. Although it was reported that proteinase K digestion was required for RNA in situ hybridization19, obvious difference was not observed between telomere FISH results with and without proteinase K treatment. In addition, freeze-cracking of embryos provided an easy-to-use method for visualizing many cells simultaneously on a single focal plane for large quantitative analysis.
By using PNA probe, telomere signal was significantly increased compared with that obtained with a DNA probe. This might be due to higher binding affinity for its complementary target and its smaller size (3 repeat compared to 4 repeat). Fluorescently labeled PNA probe also directly binds to its target, minimizing subsequent steps. Strong affinity maintained in the following immunofluorescence steps makes the post-fixation unnecessary, which can avoid background noise.
However, some limitations exist in this technique. One is that the permeabilization step slightly damages the morphology at the cost of efficient probe penetration. Treatment of gonads and embryos with methanol and acetone distorted circularity of nuclei compared to untreated control. For experiments that require perfectly preserved morphology, different permeabilization method should be attempted. Another is that the telomere signal is quantified in arbitrary units. This is mainly due to variations among independent experiments. Ribosomal DNA may be considered as an internal control to normalize each sample. More useful methods can be found in reference 20.
A simple telomere FISH protocol is described here, which requires minimum steps. Many steps are reduced for analysis of large amount of embryos. However, further modification can be made for stronger signal, such as chitinase treatment for penetration, and other innovative trials. In combination with cell culture method, super-resolution imaging may be possible. This protocol may help to discover the novel telomere maintenance mechanism in C. elegans.

Telomere protects chromosomal ends from aberrant fusion and degradation. Mammalian telomere is composed of G-rich hexameric repeats, TTAGGG, and shelterin complexes. The telomere repeat sequence of the nematode is similar to those of mammals (TTAGGC). Most eukaryotes utilize telomerase to add telomere repeats to their chromosomal ends. However, 10 - 15% of cancer cells utilize telomerase independent mechanism, known as Alternative Lengthening of Telomeres (ALT)3. Previously, we reported that telomere repeats and its associated sequences, named as TALT, were amplified in the telomeres of telomerase mutant lines that survived critical sterility2.
Telomere length was measured by quantitative PCR or by Southern blot, which provides average length of total telomeres4,5,6,7. Read count of telomere repeat in whole genome sequencing data is also an indicator of total telomere contents8. Although Single TElomere Length Analysis (STELA) could provide the length of a single telomere, it cannot provide spatial information of telomeres9. While POT-1::mCherry reporter protein provides the spatial information of telomeres in vivo, it cannot represent lengths of double-stranded telomeres, as POT-1 is a single-strand telomere binding protein10.
While aforementioned methods provide the averaged information of repetitive sequences, fluorescence in situ hybridization (FISH) allows to observe the amount and spatial pattern of individual sequences of interest on a chromosomal scale. Instead of purification of DNA, tissues or cells are fixed to preserve the native spatial information in FISH. Thus, FISH is a both quantitative and qualitative tool for observation of individual repeat sequences, such as telomere repeats.
This protocol provides an efficient method for simultaneous detection of both telomere and other repeats based on improvements from previously described methods 11,12. C. elegans larvae or adults are multicellular organism with highly differentiated cells. The heterogeneity of cells impedes on the quantitative analysis of a large number of telomere spots. To maximize the number of cells analyzed, embryos are isolated and spread on the polylysine-coated slides for FISH. In addition, this protocol can also be combined with immunofluorescence.
As a proof that the protocol works, we show that it is possible to observe and quantify two different repetitive sequences. DNA probe against TALT1 was generated with simple PCR incorporating digoxigenin-dUTP. Then this TALT1 probe and fluorescence-labeled telomere PNA probe were hybridized simultaneously. Subsequently, digoxigenin was detected by canonical immunofluorescence methods. We present here the representative images where TALT1 colocalized with the telomere in trt-1 survivors.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PNA probe</td>
			<td>PANAGENE</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Digoxigenin-Fluorescein, Fab fragments</td>
			<td>Roche</td>
			<td>11207741910</td>
			<td>use 1:200 diluted in PBST</td>
		</tr>
		<tr>
			<td>Digoxigenin-dUTP</td>
			<td>Roche</td>
			<td>11573152910</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>SIGMA-ALDRICH</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-6148</td>
			<td>prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.</td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridizaiton solution</td>
			<td></td>
			<td></td>
			<td>3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA</td>
		</tr>
		<tr>
			<td>Hybridizaiton wash solution</td>
			<td></td>
			<td></td>
			<td>2X SSC, 50% formamide</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>BIONEER</td>
			<td>C-9012</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Carlo Erba</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>SIGMA-ALDRICH</td>
			<td>H3393</td>
			<td>make 10 mg/ml for stock solution</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>67578</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO</td>
		</tr>
		<tr>
			<td>PBST</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.1% tween-20</td>
		</tr>
		<tr>
			<td>Polysorbate 20</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-2287</td>
			<td>Commercial name is Tween-20</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution (0.1 % w/v)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-8920</td>
			<td>prepare fresh 0.01 % w/v solution before use</td>
		</tr>
		<tr>
			<td>M9</td>
			<td></td>
			<td></td>
			<td>3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L</td>
		</tr>
		<tr>
			<td>Bleaching solution</td>
			<td></td>
			<td></td>
			<td>20% sodium hypochlorite, 0.5 M KOH</td>
		</tr>
		<tr>
			<td>Antibody buffer</td>
			<td></td>
			<td></td>
			<td>1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Antibody buffer with 5% bovine serum albumin (BSA)</td>
		</tr>
		<tr>
			<td>illustra Microspin G-50</td>
			<td>GE healthcare</td>
			<td>27-53310-01</td>
			<td></td>
		</tr>
		<tr>
			<td>20X SSC</td>
			<td></td>
			<td></td>
			<td>To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1 % tween-20</td>
		</tr>
		<tr>
			<td>10x digoxigenin-dUTP mix</td>
			<td></td>
			<td></td>
			<td>1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP</td>
		</tr>
		<tr>
			<td>PCR purification columns</td>
			<td>Cosmo genetech</td>
			<td>CMR0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cleaner / ULTRA CLEAN</td>
			<td>Dukssan pure chemicals</td>
			<td>8AV721</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-well glass slide</td>
			<td>MP biomedicals</td>
			<td>96041205</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode growth media</td>
			<td></td>
			<td></td>
			<td>to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4.</td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>SIGMA-ALDRICH</td>
			<td>196142</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Feather</td>
			<td>blade No. 11</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Enzynomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>SIGMA-ALDRICH</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microsope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td>EC Plan-Neofluar 100x was used as objective lens.</td>
		</tr>
		<tr>
			<td>Dry block / aluminum block</td>
			<td>Labtech</td>
			<td>LBH-T03</td>
			<td>Set temperature to 80&#8451;</td>
		</tr>
		<tr>
			<td>Humid chamber</td>
			<td></td>
			<td></td>
			<td>Plastic box filled with paper towel soaked in DW</td>
		</tr>
		<tr>
			<td>Image Analysis Software&#160;</td>
			<td>Dr. Peter Landsdorp</td>
			<td>TFL-telo</td>
			<td><a href="http://www.flintbox.com/public/project/502">http://www.flintbox.com/public/project/502</a></td>
		</tr>
	</tbody>
</table>

observation and quantification of telomere and repetitive sequences using fluorescence in situ hybridization (fish) with pna probes in caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

It was previously reported that ALT survivor can emerge from telomerase-deficient mutant, trt-1(ok410), in low frequency by replicating internally localized &#39;Template of ALT&#39; (TALT) sequences for telomere maintenance2. Using PNA probe, we were able to visualize telomeres in the dissected gonads (Figure 2A). The faint telomere signal was detected both in trt-1(ok410) and ALT survivor. The fuzzy signal was overlapped only with DAPI, suggesting that they may not be autofluorescence. Interstitial telomere-like repeat (ITR) is consistently observed in TRF assay in the study of C. elegans telomere4,10. Considering high specificity of PNA probe, they are likely to be the ITR dispersed throughout the genome.
The number of telomere spots was approximately 9 per pachytene nucleus in trt-1 (ok410). In the previous study, 12 foci was observed by POT-1::mCherry protein, which binds to single stranded telomere DNA10. Maximum of 24 foci per nucleus was observed in the wild type embryos17. The result suggests that mCherry reporter method is better for the experiment where the number of telomeres should be counted. However PNA FISH is able to detect double-stranded telomere DNA as well as single-stranded telomere DNA in proportion with the telomere length. In contrast, the number of telomere spots was approximately 7 in ALT survivor, which have fused chromosomes (N = 3) 2. This result is consistent with the prediction that telomere spots would be 6 in ALT survivor. We concluded that the signal intensity of ALT survivor was sufficient to be observed.
Telomere signal was colocalized with TALT1 in the ALT survivor, suggesting that TALT1 is used as copy template for telomere in the absence of telomerase (Figure 3). Telomere signal of ALT survivors increased compared to that of parental trt-1(ok410) mutant, indicating that telomere is robustly maintained in ALT survivors without telomerase (Figure 4). The signal of PNA probe was greater than that of digoxigenin-labeled probe (Figure 4). Designing probes with PNA oligomer might result in stronger signal than digoxigenin-labeling.
<img alt="Figure 1" src="/files/ftp_upload/54224/54224fig1.jpg" /> 
Figure 1: Overview of FISH Experiment. Eggs are harvested by bleaching adult worms and fixed in 2% PFA on a polylysine-coated slide. Samples are freeze-cracked and permeabilized with methanol and acetone for probe penetration. Probes are added to the sample and hybridized overnight at 37 &#176;C. Digoxigenin-labeled probe is detected by immunofluorescence. The samples are imaged and then quantified by the image analysis software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54224/54224fig2.jpg" /> 
Figure 2: Telomere FISH in the Dissected Gonads. (A) Telomere (red) was detected by cy3-PNA-(TTAGGC)3 in the distal tip of gonads (arrowhead). The intensity of ALT survivor is greater than that of trt-1(ok410). Z-stack image was rendered with maximum projection. Nuclei indicated by white arrow is blown-up on the upper right corner. Scale bar, 10 &#181;m. (B) Number of telomere spots per nucleus in pachytene stage was measured by visual inspection. N = 50. Error bars, SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54224/54224fig3.jpg" /> 
Figure 3: Telomere and TALT1 FISH in the Embryos. A representative image of telomere (red) and TALT1 (green) FISH. Telomere and TALT1 probe were hybridized to embryos simultaneously. DNA was counterstained with DAPI (blue). Scale bar, 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54224/54224fig4.jpg" /> 
Figure 4: Quantification of FISH Data. Telomere and TALT1 intensity from Figure 3 were quantified in the image analysis software (a gift from Dr. Peter Lansdorp). Each spot was quantified with threshold level over 15 to exclude non-specific background. T-test was used for evaluating statistical significance. (*p &#60; 0.001). Error bars, SEM. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54224/54224fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans at

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

observation-quantification-telomere-repetitive-sequences-using

Nanyang Technological University

Research

JoVE Journal

Biology

10.3K Views.  Seoul National University. We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT). 

Video: Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

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 ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

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 ...

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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.

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.