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>Transfer the slides to the sterile glass Coplin jar with 100% ethanol and soak them for 10 min. After 10 min, empty the glass Coplin jar and replace with new 100% ethanol. Soak the slides again for 10 min.</li>
		<li>Transfer the slides to the sterile glass Coplin jar with 80% ethanol for 1 min.</li>
		<li>Transfer the slides to the sterile glass Coplin jar with 70% ethanol for 1 min.</li>
		<li>Transfer the slides to the sterile glass Coplin jar with 60% ethanol for 1 min.</li>
	</ol>
	</li>
</ol>
2. Pretreatment of Slides for Preparation of RNA Probe Hybridization
<ol>
	<li>Perform the following washes at room temperature, unless specified otherwise. Perform room temperature washes on an orbital shaker at a slow speed. Use sterile slide mailers with room for up to five slides for the washing of the slides.</li>
	<li>Transfer the slides to a sterile slide mailer with 18 mL of 1x phosphate buffered saline (PBS). Wash for 5 min at room temperature.</li>
	<li>Pour off the 1x PBS, and immediately treat the tissue on the slides with proteinase K to enable probe penetration of the tissue. Add approximately 18 mL of proteinase K solution to the slide mailer (see Table 1 for working solution concentration). Incubate at 37 &#176;C for 15 min. Do not shake.
	<ol>
		<li>While the slides are incubating, prepare the prehybridization buffer (Prehybe Buffer). Place the Prehybe Buffer in a boiling water bath for 10 min, then cool on an ice bath for 5 min. For a recipe of Prehybe Buffer, see Table 1.</li>
		<li>Stop digestion of the proteinase K reaction by pouring out the proteinase K solution, and immediately add 18 mL of 0.2% glycine-PBS solution to the slide mailer. Let the slide mailer sit at room temperature for 5 min.</li>
		<li>Pour out the 0.2% glycine-PBS solution and add 18 mL of 2x saline sodium citrate (SSC) solution to each slide mailer. Wash the slides in the 2x SSC for 10 min at room temperature, with shaking at 100-150 rpm.</li>
	</ol>
	</li>
</ol>
3. Prehybridization of Slides in Preparation for RNA Probe Hybridization
<ol>
	<li>While the slides are being washed with 2x SSC, obtain a new sterile slide mailer and put approximately 18 mL of Prehybe Buffer into the slide mailer. Place the slide mailer into the hybridization oven at hybridization temperature. 
	NOTE: The hybridization temperature will depend on the probe being used but usually ranges from 50-60 &#176;C.</li>
	<li>Once the 2x SSC wash is completed, pour out the 2x SSC, and place the slides within the slide mailer in the Prehybe Buffer in the hybridization oven for 1 h.</li>
</ol>
4. Hybridization of the RNA Probe
<ol>
	<li>While the slides are incubating with the Prehybe Buffer in the hybridization oven, prepare hybridization-probe solution.
	<ol>
		<li>Dilute each probe in hybridization buffer (0.5 &#181;L of probe with 24.5 &#181;L of hybridization buffer). 
		NOTE: The recipe for hybridization buffer is found in Table 1. An example of probe preparation and concentrations can be found in Traylor-Knowles et al.11.</li>
		<li>Place the diluted probe on an 86-90 &#176;C heat block for 12 min, then cool for 1 min on ice.</li>
	</ol>
	</li>
	<li>Remove the slide mailer from the hybridization oven and individually remove slides from the slide mailer using sterile tweezers. Lay the slides flat on a paper towel, and carefully wipe off excess Prehybe Buffer around the tissue samples. Be careful not to touch the samples, and work quickly to prevent drying of the tissue samples.</li>
	<li>Encircle the tissue with a PAP pen. Apply 25 &#181;L of diluted probe solution with a pipette and cover the sample with a plastic coverslip.
	<ol>
		<li>Place the samples in the slide moisture chamber with 4x SSC + 50% formamide solution in the bottom of the chamber.</li>
		<li>Once probes have been added to all the slides, place the slide moisture chamber into the hybridization oven for at least 24 h at a hybridization temperature of 50-60 &#176;C. The incubation time can vary depending on the concentration of the probe but should be for a minimum of 24 hours.</li>
	</ol>
	</li>
	<li>After overnight incubation with the diluted probes, remove the slide moisture chamber from the hybridization oven.
	<ol>
		<li>Remove coverslips from each of the slides, being careful not to displace the tissue. In a 1000 &#181;L pipette, add 1000 &#181;L of 2x SSC solution and gently wash the slide off.</li>
		<li>Place the slide in a sterile slide mailer with 18 mL of 2x SSC solution for 5 min at room temperature. Pour out the solution and replace it with 18 mL of fresh 2x SSC. Repeat the incubation again for 5 min at room temperature with gentle shaking.</li>
		<li>Pour out the 2x SSC solution. Add 18 mL of 1x SSC solution and wash for 5 min at room temperature. Pour out the 1x SSC solution and replace it with 18 mL of fresh 1x SSC. Repeat the incubation for 5 min at room temperature with gentle shaking.</li>
		<li>Pour out the 1x SSC. Add 18 mL of 0.5x SSC and wash for 10 min at 42 &#176;C without shaking. Pour out the 0.5x SSC and replace it with 18 mL of fresh 0.5x SSC. Wash again for 10 min at 42 &#176;C without shaking.</li>
	</ol>
	</li>
</ol>
5. Visualization of the RNA Probe
NOTE: For visualizing the probe, BM purple will be used during the development process. However, before this step, several washes are required to prepare the samples for staining.
<ol>
	<li>Wash the slides with 18 mL of alkaline phosphatase buffer (AP-buffer) without MgCl2 for 1 min. Pour out the AP-Buffer without MgCl2, and add 18 mL of 1x Boehringer-Mannheim blocking buffer diluted with maleic acid buffer. Incubate for at least 1 h at room temperature in a slide mailer with gentle shaking. 
	NOTE: The Boehringer-Mannheim blocking buffer and maleic acid buffer used were premade and purchased in the DIG Wash and Block Buffer Set (available commercially).
	<ol>
		<li>Alternatively, incubation overnight at 4 &#176;C can be performed. A longer blocking period will lessen the appearance of non-specific binding.</li>
	</ol>
	</li>
	<li>Prepare the 20 mL of diluted DIG anti-digoxigenin-AP Fab fragments (anti-DIG antibody = 2 &#181;L of anti-DIG antibody + 20 mL 1x Boehringer-Mannheim blocking buffer). This is enough for use in 1 plastic slide mailer. More of this solution must be prepared if more than one slide mailer is to be used.</li>
	<li>Add the anti-DIG antibody to a new sterile slide mailer, and transfer the slides to the slide mailer with the anti-DIG antibody solution. Incubate at room temperature for 3 h with gentle shaking.</li>
	<li>After incubation, pour out the anti-DIG antibody and wash the slides in the slide mailer with 18 mL of AP-Buffer without MgCl2 for 5 min with gentle shaking.</li>
	<li>Pour out the AP-Buffer without MgCl2, and add 18 mL of AP-Buffer. Wash for 5 min with gentle shaking. After the 5 min incubation, pour off the AP-Buffer, replace it with 18 mL of fresh AP-Buffer, and wash for 5 min with gentle shaking.</li>
	<li>In the dark, pour out the AP-Buffer and add 18 mL of BM purple to the slide mailer. Incubate the slides at room temperature in the dark, checking for purple color development every &#189; h. Reaction times for visualization will vary based on the probe that is being developed. 
	NOTE: Instead of BM purple visualization, development solution can be made using 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT). See Table 1 for instructions.</li>
	<li>Stop color development by transferring the slides to a new sterile slide mailer with 18 mL of Tris-EDTA (TE) buffer for 5 min at room temperature, in the dark.</li>
	<li>Pour off the TE buffer and add 18 mL of RNase-free water to the slide mailer and wash for 1 min, in the dark.</li>
	<li>Remove the slides from the water, and dry them around the edges of the tissue. Add glycerol mounting medium and place the coverslips. Store the slides at 4 &#176;C until pictures are ready to be taken.</li>
</ol>

<ol><li>Hughes, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Climate+Change%2C+Human+Impacts%2C+and+the+Resilience+of+Coral+Reefs%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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="https://www.sciencedirect.com/science/article/pii/S0959378014001782">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/pubmed?term=((Coral+Reefs+Under+Rapid+Climate+Change+and+Ocean+Acidification%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Microarray+analysis+identifies+candidate+genes+for+key+roles+in+coral+development%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">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/pubmed?term=((The+innate+immune+repertoire+in+Cnidaria+-+ancestral+complexity+and+stochastic+gene+loss%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">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/pubmed?term=((Sox+genes+in+the+coral+Acropora+millepora%3A+divergent+expression+patterns+reflect+differences+in+developmental+mechanisms+within+the+Anthozoa%5BTitle%5D)+AND+%22BMC+Evolutionary+Biology%22%5BJournal%5D)">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/pubmed?term=((Sequence+and+expression+of+four+coral+G+protein-coupled+receptors+distinct+from+all+classifiable+members+of+the+rhodopsin+family%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">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><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22In+situ+hybridization+protocols%22">In situ hybridization protocols</a>. Darby, I. A., Hewitson, T. D. , Humuna Press. Towtowa, New Jersey. (2006).</li>
<li>Valentino, K. L., Eberwine, J. H., Barchas, J. D. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aValentino%2C+KL+%22In+situ+hybridization%22">In situ hybridization</a>. , Oxford University Press. (1987).</li>
<li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?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%5BTitle%5D)+AND+%22Chromosoma%22%5BJournal%5D)">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/pubmed?term=((The+cell+specificity+of+gene+expression+in+the+response+to+heat+stress+in+corals%5BTitle%5D)+AND+%22Journal+of+Experimental+Biology%22%5BJournal%5D)">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/pubmed?term=((Characterizing+the+spatiotemporal+expression+of+RNAs+and+proteins+in+the+starlet+sea+anemone%2C+Nematostella+vectensis%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Expression+of+multiple+Sox+genes+through+embryonic+development+in+the+ctenophore+Mnemiopsis+leidyi+is+spatially+restricted+to+zones+of+cell+proliferation%5BTitle%5D)+AND+%22EvoDevo%22%5BJournal%5D)">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/pubmed?term=((Sex-specific+and+developmental+expression+of+Dmrt+genes+in+the+starlet+sea+anemone%2C+Nematostella+vectensis%5BTitle%5D)+AND+%22EvoDevo%22%5BJournal%5D)">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/pubmed?term=((The+role+of+HoxA11+and+HoxA13+in+the+evolution+of+novel+fin+morphologies+in+a+representative+batoid+%28Leucoraja+erinacea%29%5BTitle%5D)+AND+%22EvoDevo%22%5BJournal%5D)">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/pubmed?term=((Subdivision+of+arthropod+cap-n-collar+expression+domains+is+restricted+to+Mandibulata%5BTitle%5D)+AND+%22EvoDevo%22%5BJournal%5D)">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/pubmed?term=((Cubozoan+jellyfish%3A+an+Evo%2FDevo+model+for+eyes+and+other+sensory+systems%5BTitle%5D)+AND+%22The+International+Journal+of+Developmental+Biology%22%5BJournal%5D)">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&#039;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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7.0K Views.  University of Miami, Rosenstiel School of Marine and Atmospheric Sciences. Het doel van dit protocol is voor het uitvoeren van in situ hybridisatie op volwassen koraal monsters die zijn ingesloten in paraffine en gesegmenteerd in glas dia's. Dit is een kwalitatieve methode gebruikt voor het visualiseren van de ruimtelijke uitdrukking van een sonde van de anti-zin RNA in paraffine-ingebedde weefsels.

Video: In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Neuroscience

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

Cancer Research

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and free-swimming medusae. As exemplified in established models such as Hydra and Nematostella, stem cells and/or proliferative cells contribute to the development and regeneration of cnidarian polyps. However, the underlying cellular mechanisms in most jellyfish, particularly at the medusa stage, are largely unclear, and, thus, developing a robust method for identifying specific cell types is critical. This paper describes a protocol for visualizing stem-like proliferating cells in the hydrozoan jellyfish Cladonema pacificum. Cladonema medusae possess branched tentacles that continuously grow and maintain regenerative capacity throughout their adult stage, providing a unique platform with which to study the cellular mechanisms orchestrated by proliferating and/or stem-like cells. Whole-mount fluorescent in situ hybridization (FISH) using a stem cell marker allows for the detection of stem-like cells, while pulse labeling with 5-ethynyl-2&#39;-deoxyuridine (EdU), an S phase marker, enables the identification of proliferating cells. Combining both FISH and EdU labeling, we can detect actively proliferating stem-like cells on fixed animals, and this technique can be broadly applied to other animals, including non-model jellyfish species.

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Here, we describe a protocol for visualizing stem-like proliferating cells in the jellyfish Cladonema. Whole-mount fluorescent in situ hybridization with a stem cell marker allows for the detection of stem-like cells, and 5-ethynyl-2&#39;-deoxyuridine labeling enables the identification of proliferating cells. Together, actively proliferating stem-like cells can be detected.&#160;

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and ...

Developmental Biology

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Biology

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

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.

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

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

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