Name: in situ hybridization techniques for paraffin-embedded adult coral samples
Uploaded: 2018-08-31T13:00:00
Description: Watch this Scientific Journal Video about In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples at JoVE.com

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>
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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. 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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 ...

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