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/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. 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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 samples of organisms beyond corals that have also been paraffin-embedded. Lastly, this method can be performed during different life stages of corals (e.g., larva) or within cell cultures, but the exact protocol may differ since those types of samples are not always paraffin-embedded5,6.
There are several critical steps in this protocol, including the proteinase K treatment, hybridization of the probe, and color development of the probe. During the proteinase K treatment, timing is very important. If the treatment is longer than suggested, tissues can become degraded and damaged. Additionally, the stopping of the proteinase K reaction should be performed at the full time point specified. It is also critical to keep the sample at a constant hybridization temperature, as lowering of the temperature can cause non-specific binding of the probe and complicate results. When washing off the probe, it is important to take each slide out of the hybridization oven one at a time, rather than all at once, to ensure that the slides do not hybridize with the probe at a low temperature. Also, thorough washing of the slides after the probe hybridization will help prevent non-specific binding. Lastly, color development of the probe is one of the most critical yet subjective steps of this process. The color development can be easily overdone or stopped too soon. This step requires patience and vigilance to ensure that over-staining of the slides does not occur.
Troubleshooting this protocol can be a daunting process due to the number of steps involved. If the protocol yields negative results, then it is best to start by examining the probe to make sure that it is the anti-sense (and not the sense) probe. Additionally, if the solutions are old, it is worth remaking or replacing them so they are fresh when performing this protocol. Modifications, such as the lengths of hybridization and blocking, can be implemented to increase the chances of probe hybridization or reducing amounts of background and non-specific staining. Due to the high number of steps in this protocol, it may be easy to lose track; therefore, it is important to stay organized and track which parts of the protocol have been completed. Additionally, it is critical to ensure that no steps are skipped, and that time is not cut down during the incubations and washes. A good rule of thumb is to allow for longer rather than shorter washes to ensure that all slides are cleaned thoroughly.
The biggest limitation of this method is that the results are not quantifiable. This is a qualitative method that, in concert with other methods such as histology, transcriptomics, and proteomics, is very informative. Due to variations in hybridization times as well as the subjective times during visualization, quantifying the intensity of the staining is not easily accomplished. It is tempting to say that a probe is more highly expressed if it shows greater intensity, but due to the variations in this protocol (such as the amount of time allowed for color development), gene expression amounts cannot be determined.
This technique is not a new method in biology; however, it is one not performed very often on adult corals. The use of this method on adult corals is very powerful because it anchors gene expression data to a tissue or a cell type11. Transcriptomics and genomics have become popular and powerful tools in coral biology; however, these methods are generally done on homogenates of the whole animal. This makes it challenging to identify which parts of the coral are expressing the genes of interest, and thus more challenging to understand the actual mechanisms. Through the use of ISH along with gene expression data, a more holistic approach to where and to what extent genes are expressed can be achieved. This will greatly help in the understanding of the mechanisms responsible for different stress response pathways in adult corals.

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. Figure 1A is an example of diffuse tissue staining. The stain is found throughout the tissue, not only in specific cells. Here, the expression of anti-sense AP-1 is found throughout the epidermis and the oral gastrodermis11 (Figure 1A). The staining is diffuse and not segregated to one specific cell type. For this type of staining, it is particularly critical to run a sense control at the same time, as some of the results may be due to non-specific staining.
The second type of staining is cell-specific, in which the stain is only found in specific cell types. Both FosB (Figure 1B) and TNFR41 (Figure 1C) show this type of staining. Both of these probes were used on tissue samples of adult corals that had been exposed to a heat stress11. In the anti-sense panel, purple staining is observed. TNFR41 was expressed in different types of cnidocytes, a family of cell types that is only found within cnidarians (Figure 1). Specifically, it stained nematocytes that produce the microbasic mastigophore organelle (nematocysts) and spirocytes that produce spirocysts, all found within the epidermis of coral tissue. Nematocytes are also found in other tissue layers of the coral, such as the calicodermis; however, due to the sectioning performed on this particular sample, that information could not be gathered. FosB also stained cnidocytes and was specific to both spirocytes and nematocytes. For both of these probes, the sense control showed no staining, indicating that the staining for the anti-sense probe was indeed specific. These are just two types of representative results (diffuse tissue staining and cell-specific staining) of the staining techniques that may be present with different types of probes. Due to this variability, it is critical to use a sense control to determine non-specific staining.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57853/57853fig1.jpg" /> 
Figure 1: Representative outcome of ISH that stained specific cells. (A) In the first panel, sense control for the AP-1 probe shows that little staining is present within the slide. In the second panel, the anti-sense probe for AP-1 shows that staining with this probe is diffuse throughout the tissue. The stain is specific to the oral gastrodermis and epidermis. With this type of staining, it is critical to run a sense control to ensure that the observed staining is specific. (B) A sense control for the FosB probe in the first panel shows little staining present. In the second panel, the anti-sense probe for FosB shows staining specific to the spirocytes and nematocytes, with diffuse staining throughout the epidermis and oral gastrodermis. (C) In the first panel, the sense control for the TNFR 41 probe shows that little staining is present on the slide. In the second panel, the anti-sense probe for TNFR 41 shows specific staining within the spirocytes, nematocytes, and microblastic mastigophores. Abbreviations: spirocytes (SP), symbiodium (SY), nematocyte (NE), microbasic mastigophores (MM), symbiont-containing gastrodermal cell (SU). This figure was reproduced with permission11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57853/57853fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Note: All dilutions should be done with sterile, RNase Free water in autoclaved glassware.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Prehybe Buffer (50 mL)</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">Formamide</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td colspan="3">20X SSC (pH 4.5)</td>
			<td>12.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">20 mg/mL heparin</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td colspan="3">50X Denhardts</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td colspan="3">20% Tween-20</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">20% SDS</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">10 mg/mL Salmon Sperm DNA (denature before adding)</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td colspan="3">RNase Free water</td>
			<td>4.4 mL</td>
		</tr>
		<tr>
			<td colspan="3">Note: Solution can be made in a disposible 50 mL tube. Salmon Sperm should be denatured by boiling on a heat block for 5 minutes before adding to solution.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Hybridization Buffer (47 mL) </td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">Formamide</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td colspan="3">20X SSC (pH 4.5)</td>
			<td>12.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">20 mg/mL heparin</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td colspan="3">50X Denhardts</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td colspan="3">20% Tween-20</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">20% SDS</td>
			<td>0.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">10 mg/mL Salmon Sperm DNA (denature before adding)</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td colspan="3">RNase Free water</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td colspan="3">Note: Solution can be made in a disposible 50 mL tube. Salmon Sperm should be denatured by boiling on a heat block for 5 minutes before adding to solution.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">10X PBS (1.0 L)</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">18.6 mM NaH2PO4</td>
			<td>2.56 g</td>
		</tr>
		<tr>
			<td colspan="3">84.1 mM Na2HPO4</td>
			<td>11.94 g</td>
		</tr>
		<tr>
			<td colspan="3">1,750 mM NaCl</td>
			<td>102.2 g</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Mix phosphates in about 800 mL of water for a 1.0 L volume. Check pH. It should be 7.4 &#177; 0.4. Otherwise adjust pH to 7.4 with NaOH of HCl. Add NaCl and rest of water. After pH is adjusted autoclave.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">20X SSC (1.0 L)</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">3 M NaCl</td>
			<td>175.3 g</td>
		</tr>
		<tr>
			<td colspan="3">0.3 M Na Citrate</td>
			<td>88.2 g</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Mix in about 800 mL of RNase free water to bring to a 1.0 L volume. Adjust pH to 4.5 and autoclave.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Alkaline Phosphatase (AP) buffer w/o MgCl2 (50 mL) </td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">1 M NaCl</td>
			<td>5.0 mL</td>
		</tr>
		<tr>
			<td colspan="3">1 M Tris, pH 9.5</td>
			<td>5.0 mL</td>
		</tr>
		<tr>
			<td colspan="3">20% Tween-20</td>
			<td>1.25 mL</td>
		</tr>
		<tr>
			<td colspan="3">RNase Free water</td>
			<td>38.75 mL</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Prepare just prior to use in a 50 mL tube.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Alkaline Phosphatase (AP) buffer (100 mL) </td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">1 M NaCl</td>
			<td>10.0 mL</td>
		</tr>
		<tr>
			<td colspan="3">1 M MgCl2</td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td colspan="3">1 M Tris, pH 9.5</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td colspan="3">20% Tween-20</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">RNase Free water</td>
			<td>72.5 mL</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Prepare just prior to use in a 50 mL tube. The solution will become cloudy after a few hours and will no longer work for the enzymatic reaction.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">AP Substrate Solution</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">AP Buffer</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td colspan="3">NBT</td>
			<td>82.5 uL</td>
		</tr>
		<tr>
			<td colspan="3">BCIP</td>
			<td>82.5 uL</td>
		</tr>
		<tr>
			<td colspan="3">Notes: This can be used instead of BM Purple. Prepare just prior to use in a 50 mL tube. Keep this solution in the dark by covering tube in foil, and preparing in low light.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Proteinase K Stock Solution (10 mg/mL)</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">Proteinase K</td>
			<td>10 mg</td>
		</tr>
		<tr>
			<td colspan="3">RNase Free water</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Aliquot and store at -20 &#176;C.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Proteinase K Working Solution</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">Proteinase K stock solution</td>
			<td>90 ul</td>
		</tr>
		<tr>
			<td colspan="3">1X PBS</td>
			<td>18 mL</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Make just prior to use for best results.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">0.2% glycine-PBS solution</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">10X PBS</td>
			<td>45 mL</td>
		</tr>
		<tr>
			<td colspan="3">RNase Free Water</td>
			<td>405 mL</td>
		</tr>
		<tr>
			<td colspan="3">Glycine</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Prepare in autoclaved glass container. Mix at room temperature on a stirer plate until glycine is fully dissolved.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Boehringer-Mannheim Blocking Buffer (30 mL) (Part of the DIG Wash and Block Buffer Set)</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">10x Blocking solution</td>
			<td>3 mL</td>
		</tr>
		<tr>
			<td colspan="3">1x maleic acid buffer</td>
			<td>27 mL</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Make just prior to use for best results. Stock Blocking solution and maleic acid buffer were used from the &#8220;DIG Wash and Block -Buffer Set&#34;.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">4X SSC&#8212;50% formamide (30 mL)</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">20X SSC</td>
			<td>6 mL</td>
		</tr>
		<tr>
			<td colspan="3">50% formamide</td>
			<td>24 mL</td>
		</tr>
		<tr>
			<td colspan="3">Notes: Prepare just prior to use in 50 mL tube. Prepare under laboratory hood.</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Glycerol Mounting Media</td>
			<td>ADD</td>
		</tr>
		<tr>
			<td colspan="3">50 mM Tris, pH 8.4</td>
			<td>80 &#181;L</td>
		</tr>
		<tr>
			<td colspan="3">Glycerol</td>
			<td>20 &#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Stock solutions for performing in situ hybridization. The table is a list of the different stock solutions needed for this protocol. Total amounts are estimated for performance of approximately 2 slide mailers. It is advised to adjust total amounts based on the amount of slides that are being processed.

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 ...

in-situ-hybridization-techniques-for-paraffin-embedded-adult-coral

Nanyang Technological University

Research

JoVE Journal

Environment

7.0K Views.  University of Miami, Rosenstiel School of Marine and Atmospheric Sciences. 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.

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

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

Techniques for Investigating the Anatomy of the Ant Visual System

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye anatomy of insects. These include traditional histological techniques optimized for work on ant eyes and adapted to work in concert with other techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM). These techniques, although vastly useful, can be difficult for the novice microscopist, so great emphasis has been placed in this article on troubleshooting and optimization for different specimens. We provide information on imaging techniques for the entire specimen (photo-microscopy and SEM) and discuss their advantages and disadvantages. We highlight the technique used in determining lens diameters for the entire eye and discuss new techniques for improvement. Lastly, we discuss techniques involved in preparing samples for LM and TEM, sectioning, staining, and imaging these samples. We discuss the hurdles that one might come across when preparing samples and how best to navigate around them.

This article outlines a suite of techniques in light and electron microscopy to study the internal and external eye anatomy of insects. These include several traditional techniques optimized for work on ant eyes, detailed troubleshooting, and suggestions for optimization for different specimens and regions of interest.

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Laser-Induced Fluorescence Emission (L.I.F.E.) as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about microbial-mediated carbon fluxes in extreme environments. Hence, the methodology of sample acquisition described in the very few studies available implies two major problems: A) high resolution data require a large number of samples, which is difficult to obtain in remote areas; B) unavoidable sample manipulation such as cutting, sawing, and melting of ice cores that leads to a misunderstanding of in situ conditions. In this study, a prototype device that requires neither sample preparation nor sample destruction is presented. The device can be used for in situ measurements with a high spectral and spatial resolution in terrestrial and ice ecosystems and is based on the Laser-Induced Fluorescence Emission (L.I.F.E.) technique. Photoautotrophic supraglacial communities can be identified by the detection of L.I.F.E. signatures in photopigments. The L.I.F.E. instrument calibration for the porphyrin derivates chlorophylla (chla) (405 nm laser excitation) and B-phycoerythrin (B-PE) (532 nm laser excitation) is demonstrated. For the validation of this methodology, L.I.F.E. data were ratified by a conventional method for chla quantification that involved pigment extraction and subsequent absorption spectroscopy. The prototype applicability in the field was proven in extreme polar environments. Further testing on terrestrial habitats took place during Mars analog simulations in the Moroccan dessert and on an Austrian rock glacier. The L.I.F.E. instrument enables high resolution scans of large areas with acceptable operation logistics and contributes to a better understanding of the ecological potential of supraglacial communities in the context of global change.

Laser-Induced Fluorescence Emission L.I.F.E. as Novel Non-Invasive Tool for In-Situ Measurements of Biomarkers in Cryospheric Habitats

Carbon fluxes in the cryosphere are hardly assessed yet but are crucial regarding climate change. Here we show a novel prototype device that captures the phototrophic potential in supraglacial environments based on laser-induced fluorescence emission (L.I.F.E.) technology offering high spectral and spatial resolution data under in situ conditions.

Global warming affects microbial communities in a variety of ecosystems, especially cryospheric habitats. However, little is known about ...

In Situ Chemotaxis Assay to Examine Microbial Behavior in Aquatic Ecosystems

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread across the bacterial and archaeal domains. Chemotaxis can result in substantial resource acquisition advantages in heterogeneous environments. It also plays a crucial role in symbiotic interactions, disease, and global processes, such as biogeochemical cycling. However, current techniques restrict chemotaxis research to the laboratory and are not easily applicable in the field. Presented here is a step-by-step protocol for the deployment of the in situ chemotaxis assay (ISCA), a device that enables robust interrogation of microbial chemotaxis directly in the natural environment. The ISCA is a microfluidic device consisting of a 20 well array, in which chemicals of interest can be loaded. Once deployed in aqueous environments, chemicals diffuse out of the wells, creating concentration gradients that microbes sense and respond to by swimming into the wells via chemotaxis. The well contents can then be sampled and used to (1) quantify strength of the chemotactic responses to specific compounds through flow cytometry, (2) isolate and culture responsive microorganisms, and (3) characterize the identity and genomic potential of the responding populations through molecular techniques. The ISCA is a flexible platform that can be deployed in any system with an aqueous phase, including marine, freshwater, and soil environments.

Presented here is the protocol for an in situ chemotaxis assay, a recently developed microfluidic device that enables studies of microbial behavior directly in the environment.

Microbial behaviors, such as motility and chemotaxis (the ability of a cell to alter its movement in response to a chemical gradient), are widespread ...