This work was supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 31801034), the Natural Science Foundation of Fujian Province, China (2016J01161), the Scientific Research Funds of Huaqiao University (15BS306) and Postgraduates&#8217; Innovative Fund in Scientific Research of Huaqiao University.

The animal procedure was approved by the Animal Care and Welfare Committee of Huaqiao University.
1. Riboprobe preparation
<ol>
	<li>Primer design
	<ol>
		<li>Open the program Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). Copy the dmrt1 sequence (GenBank: MK182259) into the sequence window.</li>
		<li>Set primer length (23-25 bp), melting temperature (55-60 &#176;C), and G/C content (40-60%). Click Pick Primers. 
		NOTE: The minimal size required for the RNA probe is approximately 500 bp. Longer probes normally have higher specificity.</li>
		<li>Add the T7 RNA polymerase promoter sequence (5, -TAATACGACTCACTATAGGG-3,) to one of the selected primers. The dmrt1 primer sequences for ISH are shown in Table 1. 
		NOTE: For sense probes, the T7 RNA polymerase promoter is located at the 5&#8217; end of the forward primers. For antisense probes, the T7 RNA polymerase promoter is located at the 5&#8217;-end of the reverse primers.</li>
	</ol>
	</li>
	<li>PCR amplification
	<ol>
		<li>Place the microfuge tubes on ice, and prepare the following reaction mix (for a 50 &#181;L reaction): 1x &#160;Taq DNA polymerase mix, 1 &#181;g of cDNA (which is prepared from 100 &#181;L of coelomic fluid), 1 &#181;M forward primer, 1 &#181;M reverse primer and nuclease-free water.</li>
		<li>Mix the PCR reaction by pipetting and centrifuge briefly.</li>
		<li>Place the reaction tube in a thermal cycler, and run the PCR using the following conditions: initial denaturation at 95 &#176;C for 2 min followed by 36 cycles of denaturation at 95 &#176;C for 30 s, annealing at 55-60 &#176;C for 30 s, extension at 72 &#176;C for 30 s and a final extension at 72 &#176;C for 7 min. 
		NOTE: The annealing temperature should be optimized according to the primers.</li>
	</ol>
	</li>
	<li>PCR product purification and verification
	<ol>
		<li>Load the 50 &#181;L of PCR mixture directly on a 1% agarose gel. Run the agarose gel at 150-180 V for 10 min in 0.5x TBE. Isolate the specific DNA fragments from the 1% agarose gel. 
		NOTE: DNA should appear as a single band and not as a smear.</li>
		<li>Purify the DNA fragments using a gel extraction kit according to the manufacturer&#39;s protocol. Quantify the purified products by spectrophotometry at a wavelength of 260 nm.</li>
		<li>Verify the authenticity of the PCR products by sequencing. 
		NOTE: (Pause point) The purified PCR products can be stored at -20 &#176;C for several months.</li>
	</ol>
	</li>
	<li>Riboprobe synthesis
	<ol>
		<li>Place the RNase-free microfuge tubes on ice, and add the following to the microfuge tube (for a 10 &#181;L reaction): 1x Digoxygenin RNA Labeling Mix, 1x transcription buffer, 0.5 &#181;L of RNase inhibitors, 1 &#181;L of T7 RNA polymerases, 1 &#181;g of PCR product and RNase-free water. Mix the reaction by pipetting and centrifuge briefly. Incubate for 2 h at 37 &#176;C.</li>
		<li>Add 2 &#181;L of RNase-free DNase I. Mix the reaction by pipetting and centrifuge briefly. Incubate for 15 min at 37 &#176;C.</li>
		<li>Add 2 &#181;L 0.2 M EDTA (pH 8.0). Mix the reaction by pipetting and centrifuge briefly.</li>
		<li>Add 2.5 &#181;L of 4 M LiCl and 75 &#181;L of prechilled ethanol to the above reaction. Mix well. Leave for 30 min at -70 &#176;C.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C. Decant the ethanol. Add 1 mL of prechilled 70% ethanol (v/v) and wash the precipitation by mixing gently.</li>
		<li>Centrifuge at 12,000 x g for 5 min at 4 &#176;C. Decant the 70% ethanol and dry the precipitation briefly near an alcohol lamp. Dissolve the precipitation by adding 30 &#181;L of RNase-free water and mix gently.</li>
		<li>Load 2 &#181;L of synthesized RNA on a 1% agarose gel. Run the agarose gel at 180 V for 5-10 min in 0.5x TBE. Measure the concentration of the labeled RNA using a spectrophotometer at a wavelength of 260 nm.
		<ol>
			<li>Use RNase-free microfuge tubes and filter tips to avoid RNase contamination. 
			NOTE: (Pause point) The digoxygenin-labeled probes can be stored at -70 &#176;C for several months.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Coelomic fluid collection
<ol>
	<li>Fix the S. nudus on the dissection table with pins. Open the body of the S. nudus with small autoclaved scissors. Isolate the coelomic fluid with a pipette.</li>
	<li>Collect and transfer 1 mL coelomic fluid with a pipette to poly-D-lysine treated microscope slides. Apply the coelomic fluid evenly with a pipette tip. Air-dry the slides for 1 h at 37 &#176;C.</li>
</ol>
3. In situ hybridization
<ol>
	<li>Day 1
	<ol>
		<li>Rehydrate the slides in a slide staining jar containing 100 mL of 1x diethyl pyrocarbonate treated phosphate buffered saline (DEPC-PBS). Rehydrate 3 times with 1x DEPC-PBS, 5 min per wash with gentle agitation.</li>
		<li>Permeabilize the smear of coelomic fluid by digestion with 10 &#181;g/mL proteinase K at room temperature for 5 min. Incubate the slides in 4% paraformaldehyde (PFA) for 20 min to stop the digestion. Wash the slides in 1x DEPC-PBS with gentle agitation for 5 min 3 times.</li>
		<li>Add 50 &#181;L of hybridization mix (HM) containing the sense/antisense riboprobe on the slides and add a cover slip.</li>
		<li>Add wet box buffer into the wet box. Put the slides into the wet box and seal well with paraffin film. Hybridize overnight (at least 16 h) at 60 &#176;C.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Preheat the wash buffer at 65 &#176;C. Immerse the slide in the slide staining jar containing the wash buffer and let it stand until the coverslip slides off automatically. It takes about 5 min.</li>
		<li>Wash 2 times with wash buffer at 65 &#176;C, 30 min per wash with gentle agitation. Wash 2 times with 0.2x saline-sodium citrate (SSC) at 65 &#176;C, 30 min per wash with gentle agitation. Wash 2 times with maleic acid buffer containing Tween 20 (MABT) at room temperature, 30 min per wash with gentle agitation.</li>
		<li>Incubate the slides at room temperature for 3&#8211;4 h in the blocking buffer. Incubate the slides in 1 mL anti-digoxigenin-AP Fab fragments solution diluted at 1/5000 with the blocking buffer at 4 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Remove the antibody solution and wash the slides briefly in MABT. Wash 4 times with MABT at room temperature, 25 min per wash with gentle agitation.</li>
		<li>Incubate the slides with alkaline phosphatase buffer at room temperature 3 times, 5 min per wash. Remove the alkaline phosphatase buffer, and add 1 mL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) staining solution. Keep the slides in the dark.</li>
		<li>Observe the color reaction periodically under an optical microscope. When the color is fully developed (reaction time in the range of 1-4 h), wash the slides 2 times with MABT at room temperature, 5 min per wash with gentle agitation.</li>
		<li>Wash 2 times with stop solution at room temperature, 15 min per wash with gentle agitation. Incubate the slides in methanol to remove excess stain, at room temperature for 30 min. According to the background color, the methanol wash can be done 2 or 3 times and the decolorization time can be extended.</li>
		<li>Wash 2 times with MABT at room temperature, 5 min per wash with gentle agitation. Transfer the slides to a fresh filter paper. Add 50 &#181;L of glycerol. Add the coverslips and observe microscopically. 
		NOTE: The solution composition is showed in Table 2.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Previous studies showed that ISH is suitable for detecting multiple target RNAs16,17,18. In this protocol, we described a high-resolution ISH method to detect the mRNA in coelomic fluid and emphasize the optimized hybridization conditions in Sipunculus nudus. The obvious signals of dmrt1 we observed demonstrated the successful application of this protocol in the detection of gene expression (<strong class="xfig...

ISH, using a labeled nucleic acid probe to detect the specific DNA or RNA sequence of interest, is a useful method to describe the spatial expression pattern of target genes in morphologically preserved tissues1,2,3. Normally, the target sequence is generated by polymerase chain reaction (PCR), and then used as the template to synthesize the antisense/sense RNA probe labeled with digoxigenin uridine-5&#8217;-triphosphate. Samples are fixed and permeabilized before incubation with riboprobe. After washing off the excess probe, hybridization is visualized by immunohistochemistry using an anti-digoxygenin antibody, which is alkaline phosphatase-conjugated3,4,5,6.
Sipunculus nudus (Phylum Sipuncula; order Sipunculida: Sipunculidae) is an unsegmented, coelomate and bilaterally symmetrical marine worm7,8. Sipunculus nudus is a cosmopolitan species widely distributed in tropical and temperate coastal waters. It is also an important marine fishery resource in southern China because of its high nutritional and medicinal values9,10. However, Sipunculus nudus in molecular biology is still in its infancy. To fully understand the biological role of genes, the investigation of genes expression patterns at a cellular resolution is of great interest. In the non-model organism Sipunculus nudus, the ISH method, which is an ideal method to detect the genes expression patterns, has not yet been established. Its coelomic fluid contains several cell types, including granulocytes, urn cells, vesicular cells, germ cells, erythrocytes, etc11. The double sex/mab-3 related transcription factor-1 (dmrt1), used as a representative gene in this method, is a highly conserved transcriptional regulator of sex determination and differentiation in most species ranging from invertebrates to mammals12,13. In a range of species (i.e., black porgy, etc.)14, dmrt1 was expressed in the Sertoli cells, surrounding the germinal cells, whose function is similar to trophoblast cells of Sipunculus nudus. Therefore, we hypothesized that dmrt1 of Sipunculus nudus is expressed in trophoblast cells of spermatozeugmata, and the result of the ISH method clearly confirmed the hypothesis.
This protocol for the first time describes ISH, with digoxigenin-labeled antisense/sense mRNA probes, to determine mRNA expression patterns in its coelomic fluid smear. The optimal reaction conditions are supplied, which allow very sensitive visualization of mRNA expression at high resolution. The developed ISH method could be potentially applied in more Sipunculida species other than Sipunculus nudus.

<table border="0" cellpadding="0" cellspacing="0" style="width:704px;" width="703">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Agarose</td>
			<td style="width:121px;">Biowest</td>
			<td style="width:161px;">111860</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-digoxigenin-AP Fab fragments</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCIP/NBT alkaline phosphatase color development kit</td>
			<td>Beyotime</td>
			<td>C3206</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>B2064</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric acid</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>5949-29-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric acid trisodium</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>4/3/6132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coverslips</td>
			<td>Beyotime</td>
			<td>FCGF60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deionized formamide</td>
			<td>Amresco</td>
			<td>12/7/1975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diethyl pyrocarbonate, DEPC</td>
			<td>Sigma</td>
			<td>D5758</td>
			<td>Noxious substance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digoxigenin (DIG) RNA Labeling Mix</td>
			<td>Roche</td>
			<td>11277073910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I, RNase-free</td>
			<td>Invitrogen</td>
			<td>18047019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Sigma</td>
			<td>431788</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrophoresis gel imaging</td>
			<td>Bio-Rad</td>
			<td>Universal Hood III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel extraction kits</td>
			<td>Omega</td>
			<td>D2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel-electrophoresis apparatus</td>
			<td>Beijing Liuyi Instrument Factory</td>
			<td>DYY-6C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma</td>
			<td>G5417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Sigma</td>
			<td>8/1/9041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LiCl</td>
			<td>Sigma</td>
			<td>203637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maleic acid</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>110-16-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>67-56-1</td>
			<td>Noxious substance</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgCl2</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Biofount</td>
			<td>JT0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Sinopharm Chemical Reagent Co.</td>
			<td>1310-73-2</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraffin film</td>
			<td>Bemis Company, Inc.</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde, PFA</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>Noxious substance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Instrument</td>
			<td>Life Technology</td>
			<td>Proflex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pins</td>
			<td>Deli</td>
			<td>16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette</td>
			<td>Eppendorf</td>
			<td>plus G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine treated microscope slides</td>
			<td>Liusheng</td>
			<td>VER_A01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Sigma</td>
			<td>SRE0005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase free water</td>
			<td>HyClone</td>
			<td>SH30538. 02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase inhibitor</td>
			<td>Roche</td>
			<td>3335399001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S. nudus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sheep serum</td>
			<td>Beyotime</td>
			<td>C0265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide staining jar</td>
			<td>Beyotime</td>
			<td>FG010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide storage box</td>
			<td>Beyotime</td>
			<td>FBX011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small autoclaved scissors</td>
			<td>Shuanglu</td>
			<td>sku_8330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrophotometers</td>
			<td>Thermo Fisher</td>
			<td>NanoDrop 2000/2000c</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 RNA polymerases</td>
			<td>Roche</td>
			<td>10881767001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq DNA polymerase mix (2X)</td>
			<td>Thermo Scientific</td>
			<td>K1081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl</td>
			<td>Sigma</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tRNA</td>
			<td>Roche</td>
			<td>10109517001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td>Zhengzhou Great Wall Scientific Industrial</td>
			<td>HH-S2</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ hybridization for sipunculus nudus coelomic fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

A summary of the steps involved in ISH is illustrated in Figure 1. Antisense and corresponding sense riboprobes for dmrt1 were synthesized from PCR products amplified from the coelomic fluid cDNAs. The authenticity of the PCR products was verified by direct sequencing. Riboprobes were synthesized using T7 RNA polymerases according to the manufacturer&#39;s protocols and a previous report4 with some minor modifications. The representative signals of ISH are sh...

Watch this Scientific Journal Video about In situ Hybridization for Sipunculus nudus Coelomic Fluid at JoVE.com

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

in-situ-hybridization-for-sipunculus-nudus-coelomic-fluid

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Biology

5.3K Views.  Huaqiao University. This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

Video: In situ Hybridization for Sipunculus nudus Coelomic Fluid

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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

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

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

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

Fluorescence in situ hybridization FISH Protocol in Human Sperm

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

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

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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 ...

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

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

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

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

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

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

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

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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