This work was funded by award no. OCE-1323652 through the National Science Foundation Ocean Science Postdoctoral Fellowship and award no.1012629 from the Burroughs Wellcome Fund Postdoctoral Enrichment Program.

1. Removal of Paraffin
Caution: Perform the following steps under a fume hood.
<ol>
	<li>Dewax the thin-sectioned paraffin-embedded slides with 100% xylene under the hood in glass Coplin jars for 10 min. Do not use plastic Coplin jars, as xylene melts plastic. Sterilize the Coplin jars in an autoclave before use.</li>
	<li>Prepare four sterile glass Coplin jars with the following: 100% ethanol, 80% ethanol, 70% ethanol and 60% ethanol. Dilute the ethanol with RNase-free water.
	<ol>
		<li>Tr...

<ol>
	<li>Hughes, T. P., 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=Climate+Change,+Human+Impacts,+and+the+Resilience+of+Coral+Reefs.">Climate Change, Human Impacts, and the Resilience of Coral Reefs.</a> Science. 301 (5635), 933 (2003).</li><li>Chen, P. -. Y., Chen, C. -. C., Chu, L., McCarl, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+economic+damage+of+climate+change+on+global+coral+reefs.">Evaluating the economic damage of climate change on global coral reefs.</a> Global Environmental Change. 30 (Supplement C), 12-20 (2015).</li><li>Hoegh-Guldberg, O., 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=Coral+Reefs+Under+Rapid+Climate+Change+and+Ocean+Acidification.">Coral Reefs Under Rapid Climate Change and Ocean Acidification.</a> Science. 318 (5857), 1742 (2007).</li><li>Grasso, L. C., 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=Microarray+analysis+identifies+candidate+genes+for+key+roles+in+coral+development.">Microarray analysis identifies candidate genes for key roles in coral development.</a> BMC Genomics. 9 (1), 540 (2008).</li><li>Miller, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+innate+immune+repertoire+in+Cnidaria+-+ancestral+complexity+and+stochastic+gene+loss.">The innate immune repertoire in Cnidaria - ancestral complexity and stochastic gene loss.</a> Genome Biology. 8 (4), R59 (2007).</li><li>Shinzato, C., Iguchi, A., Hayward, D. C., Technau, U., Ball, E. E., Miller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox+genes+in+the+coral+Acropora+millepora:+divergent+expression+patterns+reflect+differences+in+developmental+mechanisms+within+the+Anthozoa.">Sox genes in the coral Acropora millepora: divergent expression patterns reflect differences in developmental mechanisms within the Anthozoa.</a> BMC Evolutionary Biology. 8 (1), 311 (2008).</li><li>Anctil, M., Hayward, D. C., Miller, D. J., Ball, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+and+expression+of+four+coral+G+protein-coupled+receptors+distinct+from+all+classifiable+members+of+the+rhodopsin+family.">Sequence and expression of four coral G protein-coupled receptors distinct from all classifiable members of the rhodopsin family.</a> Gene. 392 (1-2), 14-21 (2007).</li><li>Darby, I. A., Hewitson, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization protocols. , (2006).</li><li>Valentino, K. L., Eberwine, J. H., Barchas, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In situ hybridization. , (1987).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98 (2), 81-85 (1989).</li><li>Traylor-Knowles, N., Rose, N. H., Palumbi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+specificity+of+gene+expression+in+the+response+to+heat+stress+in+corals.">The cell specificity of gene expression in the response to heat stress in corals.</a> Journal of Experimental Biology. 220 (10), (2017).</li><li>Wolenski, F. S., Layden, M. J., Martindale, M. Q., Gilmore, T. D., Finnerty, 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=Characterizing+the+spatiotemporal+expression+of+RNAs+and+proteins+in+the+starlet+sea+anemone,+Nematostella+vectensis.">Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis.</a> Nature Protocols. 8, 900 (2013).</li><li>Schnitzler, C. E., Simmons, D. K., Pang, K., Martindale, M. Q., Baxevanis, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+multiple+Sox+genes+through+embryonic+development+in+the+ctenophore+Mnemiopsis+leidyi+is+spatially+restricted+to+zones+of+cell+proliferation.">Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation.</a> EvoDevo. 5 (1), 15 (2014).</li><li>Traylor-Knowles, N. G., Kane, E. G., Sombatsaphay, V., Finnerty, J. R., Reitzel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+and+developmental+expression+of+Dmrt+genes+in+the+starlet+sea+anemone,+Nematostella+vectensis.">Sex-specific and developmental expression of Dmrt genes in the starlet sea anemone, Nematostella vectensis.</a> EvoDevo. 6 (1), (2015).</li><li>Barry, S. N., Crow, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+HoxA11+and+HoxA13+in+the+evolution+of+novel+fin+morphologies+in+a+representative+batoid+(Leucoraja+erinacea).">The role of HoxA11 and HoxA13 in the evolution of novel fin morphologies in a representative batoid (Leucoraja erinacea).</a> EvoDevo. 8 (1), 24 (2017).</li><li>Sharma, P. P., Gupta, T., Schwager, E. E., Wheeler, W. C., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdivision+of+arthropod+cap-n-collar+expression+domains+is+restricted+to+Mandibulata.">Subdivision of arthropod cap-n-collar expression domains is restricted to Mandibulata.</a> EvoDevo. 5 (1), 3 (2014).</li><li>Piatigorsky, J., Kozmik, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cubozoan+jellyfish:+an+Evo/Devo+model+for+eyes+and+other+sensory+systems.">Cubozoan jellyfish: an Evo/Devo model for eyes and other sensory systems.</a> The International Journal of Developmental Biology. 48 (8-9), 719-729 (2004).</li></ol>

The authors have nothing to disclose.

The method described in this protocol has been modified from previous work in medical and evolutionary developmental research8,9,10,12,17. This protocol focuses on the nuances of an in situ hybridization with a DIG-labeled RNA anti-sense probe on adult corals, which have been preserved and embedded in paraffin. This method can be easily transferred to...

Corals are critical ecosystem builders and important for biodiversity in ocean and human health1,2,3. They are under threat due to climate change and other anthropogenic stressors, and many coral species are considered critically endangered. Thus, there is a significant need for cellular and molecular tools to diagnose corals under stress. Also, there is little understood about where genes are expressed within adult coral tissue, and therefore little understanding of the functions of these genes. To address this issue, we have adapted the in situ hybridization (ISH) protocol, commonly used in human medicine and evolutionary developmental biology, for use on paraffin-embedded tissue samples of adult corals. This technique is most powerful when used on adult corals that have undergone a stressful event such as exposure to heat stress. However, this technique can be used on a wide range of tissues and life stages in corals and is not limited to only heat-stressed corals4,6,7. Additionally, this technique can be used on tissues or cells of any metazoan as long as there is cDNA sequence information available.
The purpose of this method is to visualize RNA probes within adult coral tissue that has been preserved and embedded in paraffin and sectioned onto slides. This method is a powerful diagnostic tool that allows for the visualization of nucleic acids within adult coral tissue. Initially this method was developed for medical diagnostics, and it has since become a popular tool in fields such as developmental biology and evolutionary developmental biology8,9,10. ISH is also a critical method, particularly in non-model systems, when genomic and transcriptomic sequence data are available but spatial gene expression patterns are unknown. For diagnostic work in non-model systems, this technique is powerful because it can indicate which cells and tissues express a gene of interest and can lead to more targeted therapeutic approaches8,9,10,11,12. Lastly, this technique is qualitative and more powerful when paired with quantitative gene expression data11.
The approach outlined in this paper will be of interest to researchers who have already designed a digoxigenin (DIG)-labeled RNA probe (both the sense and antisense probes) and are now ready to perform in situ hybridization of the probes to a sample. To perform this method, two serial sections of a paraffin coral tissue will be needed for each probe being tested. One section will be used for the sense probe and the other for the antisense probe. The sense probe will be a control to indicate non-specific binding. If staining is observed in the sense probe, then the antisense probe is not specific to the RNA of interest. Probes can be designed for any gene expressed. In this protocol, several examples are used that were previously found to be expressed during heat stress in corals: FBJ murine osteosarcoma viral oncogene homolog B (Fos-B), Activator protein (AP1), and Tumor necrosis factor receptor 41 (TNFR 41)11. ISH using DIG-labeled RNA probes is preferred over using radioactive probes because their handling is much safer10. In addition, this technique is highly sensitive and can be performed on a wide range of tissues and embryos beyond heat-stressed adult corals13,14,15,16.

<table>
	<tbody>
		<tr>
			<td>Denhardt&#39;s solution</td>
			<td>Affymetrix</td>
			<td>70468 50 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioworld Alkaline phosphatase buffer</td>
			<td>Fisher</td>
			<td>50-198-724</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide mailers</td>
			<td>Fisher</td>
			<td>12-587-17B</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioworld Alkaline phosphatase buffer</td>
			<td>Fisher</td>
			<td>50-198-724</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>Fisher</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Salmon Sperm DNA solution</td>
			<td>Invitrogen</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS - Phosphate-Buffered Saline (10X) pH 7.4</td>
			<td>Invitrogen</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water, 10 x 500 mL</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure DNase/RNase-Free Distilled Water, 10 x 500 mL</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Salmon Sperm DNA solution</td>
			<td>Invitrogen</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide white apex superior adhesive</td>
			<td>Leica Biosystems</td>
			<td>3800080</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS solution, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010072</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, Molecular Grade, 2 mL</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Pap Pen Liquid Blocker</td>
			<td>Promega</td>
			<td>22309</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG Anti-Digoxigenin-AP Fab fragments</td>
			<td>Roche</td>
			<td>11093274910</td>
			<td></td>
		</tr>
		<tr>
			<td>BM Purple, 100 mL</td>
			<td>Roche</td>
			<td>11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG Wash and Block Buffer Set</td>
			<td>Roche</td>
			<td>11585762001</td>
			<td></td>
		</tr>
		<tr>
			<td>NBT/BCIP</td>
			<td>Roche</td>
			<td>11681451001</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution, 500 mL size</td>
			<td>Sigma-Aldrich</td>
			<td>252549-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC Buffer 20X concentration</td>
			<td>Sigma-Aldrich</td>
			<td>S6639-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>320102-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma-Aldrich</td>
			<td>47670-250ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>90279-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, AR (ACS), For Histological Use</td>
			<td>VWR</td>
			<td>MK866806</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>EM-EX0276-4S</td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td>VWR</td>
			<td>PAV6232</td>
			<td></td>
		</tr>
		<tr>
			<td>hybridization oven</td>
			<td>VWR</td>
			<td>97005-252, 97005-254</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-088</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ hybridization techniques for paraffin-embedded adult coral samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

After completing this protocol, identification of cells and tissues that are expressing the RNA probe of interest will be achieved. The representative results for this protocol are for AP-1, FosB, and TNFR41. These results, previously published by Traylor-Knowles et al.11, show spatial expression of RNA probes on adult corals that were exposed to heat stress. Two examples of different staining types are presented in Figure 1. ...

Watch this Scientific Journal Video about In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples at JoVE.com

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

This method can help answer key questions in coral cell biology, such as what types of tissues and cells express specific types of genes. The main advantage of this technique is that you have visual information on RNA expression. Though this method can provide insight into spatial gene expression in adult corals, it can also be used in other life stages of corals, as well as in other invertebrate systems.Generally individuals new to this method will struggle because there's so many steps and solutions to keep track of. So they need to pay attention to where they are in the protocol, and keep good track of time when doing the washes. Kevin Rodriguez, a technician in my laboratory, will be demonstrating this procedure.To begin, dewax thin sectioned paraffin embedded slides with 100%xylene in glass Coplin jars for 10 minutes under the hood. Next, fill four sterile glass Coplin jars with 100%ethanol, 80%ethanol, 70%ethanol and 60%ethanol respectively. First, transfer the slides to the Coplin jar containing 100%ethanol, and allow them to soak for 10 minutes.Then transfer the slides to another Coplin jar containing 100%ethanol. Repeat this process with each of the Coplin jars in decreasing order of ethanol concentration. First transfer the slides to a sterile slide mailer with 18 mL of PBS.Then wash the slides for five minutes at room temperature. Next, pour off 1 volume of PBS, and add approximately 10 mL of proteinase K solution to the mailer. Then incubate the mailer at 37 degrees Celsius for 15 minutes.While the slides incubate, place prehyb buffer in a boiling water bath for 10 minutes. Then allow the buffer to cool in an ice bath for 5 minutes. To stop digestion by the proteinase K reaction, remove the proteinase K and add 18 mL of 0.2%glycine PBS solution to the mailer.Then allow the mailer to sit at room temperature for 5 minutes. After this, remove the glycine PBS solution and replace it with 18 mL of 2x saline sodium citrate, or SSC. Wash the slides in SSC for 10 minutes at room temperature with shaking at 100-150 RPM.While the slides incubate in the hybridization oven, dilute the probes in hybridization buffer. Then place the diluted probes on a heat block at 86-90 degrees Celsius for 12 minutes, and allow them to cool on ice for one minute. Next, remove the slide mailer from the hybridization oven, and remove each slide with sterile tweezers.Lay the slides on a paper towel, and carefully remove excess buffer around the tissue samples. Using a PAP pen, draw a circle around each tissue sample. Then use a pipette to apply 25 microliters of diluted probe solution, and cover each sample with a plastic coverslip.After this, add 4x SSC and 50%formamide solution in the bottom of a slide moisture chamber. Then place the slides in the moisture chamber. Wash the slides with 18 mL of alkaline phosphatase buffer without magnesium chloride for 1 minute.Then pour out the buffer and replace it with 18 mL of Boehringer Mannheim blocking buffer diluted with maleic acid buffer. Incubate the slide mailer overnight at room temperature with gentle shaking. Next, prepare 20 mL of diluted DIG anti-deoxigenin-AP Fab fragments.Add the anti-dig antibody to a new sterile slide mailer, and transfer the slides from the old mailer. Then incubate the slides at room temperature for three hours with gentle shaking. After the incubation period, pour the anti-dig antibody out, and wash the slides with 18 mL of AP buffer without magnesium chloride for five minutes with gentle shaking.Next, pour out the AP buffer without magnesium chloride and add 18 mL of AP buffer. Wash the slides for five minutes with gentle shaking. Then remove the old AP buffer, replace it with 18 mL of fresh AP buffer, and repeat the wash.In a dark room, pour off the AP buffer and add 18 mL of BM-Purple to the mailer. Then incubate the slides at room temperature, checking for purple color development every half hour. To stop color development, transfer the slides to a new sterile mailer with 18 mL of TE buffer.Allow the slides to sit in the dark at room temperature for five minutes. Next, pour off the TE buffer, add 18 mL of RNase-Free water to the mailer, and wash the slides for one minute. Then remove the slides from the water and dry them with tissue.Add glycerol mounting medium, and place a coverslip on each slide. Finally, store the slides at 4 degrees Celsius until pictures are ready to be taken. Using this protocol, heat stressed coral tissue expressing AP-1, FosB, and TNFR41 were identified.As indicated by the diffuse tissue staining, the expression of anti-sense AP-1 is found throughout the epidermis and oral gastrodermis. Cell specific staining was also observed in FosB and TNFR41 expression. FosB stained cnidocytes and was specific to both spirocytes, which produce spirocysts, and nematocytes, which produce the microbasic mastigophore organelle.Likewise, TNFR41 was also expressed in nematocytes and spirocytes. While attempting this procedure, it's important to remember to not rush anything, because the washes and the incubations take time.

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6.9K Views.  University of Miami, Rosenstiel School of Marine and Atmospheric Sciences. This method can help answer key questions in coral cell biology, such as what types of tissues and cells express specific types of genes. The main advantage of this technique is that you have visual information on RNA expression. Though this method can provide insight into spatial gene expression in adult corals, it can also be used in other life stages of corals, as well as in other invertebrate systems.Generally individuals new to this method will struggle because there's so many ste...