Name: a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis
Uploaded: 2016-03-15T15:00:00.000+00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

This work was supported by funding to DJJ through DFG project #JA2108/2-1.

NOTE: The following steps outline our method for conducting an in situ experiment on embryonic and larval stages of L. stagnalis. Where a step involves the use of a hazardous chemical this is indicated by the word &#39;CAUTION&#39; and all appropriate safety procedures should be adopted. Links to representative MSDS sheets for hazardous chemicals are provided in Supplementary File 1. Recipes for all reagents are provided in Supplementary File 2.
1. Assembly of Decapsulation Apparatus
<ol>
	<li>To do this, connect a 20 ml disposable syringe to silicon tubing (with an inner diameter of 1 mm and an outer diameter of 3 mm) using a P1,000 tip cut to an appropriate length as shown in Figure&#160;2A.</li>
	<li>Tape a standard microscope slide to an inverted large Petri dish. Tape the silicon tubing immediately adjacent to the microscope slide as shown in Figure&#160;2C and 2D.</li>
	<li>Rest a pulled glass needle on the microscope slide and gently insert it into the silicon tubing until the tip of the needle protrudes approximately 20% of the way across the inner diameter of the tubing (see Figure&#160;2C and 2D). Once the needle is in position tape it down to the Petri dish.</li>
	<li>Allow the free end of the silicon tubing to rest in another Petri dish that will collect the decapsulated material.</li>
</ol>
2. Sample Collection, Fixation and Decapsulation
NOTE: All steps are carried out at RT&#160;unless otherwise noted.
<ol>
	<li>Carefully collect egg strings from the walls of an aquarium. To do this, use a flat piece of flexible plastic as a spatula to scrape the egg string off the substrate, and use a plastic tea strainer to fish the floating egg string out of the water. Stage and sort the material under a microscope using the guide provided in Figure 1.</li>
	<li>Place the egg string onto a paper towel and make a longitudinal incision along the egg mass using the featherweight forceps. Roll the egg capsules out of the egg string and remove as much of the jelly material as possible from each capsule by pushing them around the paper towel using the featherweight forceps.</li>
	<li>Using the featherweight forceps, transfer the egg capsules into a Petri dish containing 5 ml of tap water. Continue to collect de-jellied egg capsules of the desired developmental stages into this dish. Collect enough capsules from all developmental stages for the planned WMISH experiment then proceed to the next step.</li>
	<li>When working with more developed larvae (5 days post first cleavage (dpfc) and older) anaesthetize them prior to fixation. 
	NOTE: This will prevent muscles from contracting which makes the interpretation of in situ staining patterns very difficult.
	<ol>
		<li>Relax larvae (while they are still in their capsules) in a 2% w/v solution of MgCl2&#8226;6H2O for 30 min prior to fixation.</li>
		<li>Assess the degree of relaxation after 30 min by submerging several larvae while still in their egg capsules in fixative solution and monitoring their response under a microscope. Incompletely relaxed larvae will retract into their shells, while fully relaxed larvae will not respond. Once these larvae have been relaxed proceed to the next step.</li>
	</ol>
	</li>
	<li>Transfer the egg capsules using a wide bore plastic pipette into a sealable tube that provides 10 times the volume of egg capsules (e.g., 1 ml of settled capsules would require a 10+ ml tube).</li>
	<li>Aspirate as much liquid as possible from the tube and replace with a volume of fixative solution that is 10x the volume of the settled egg capsules. Gently rotate the egg capsules in fixative at RT&#160;for the appropriate time for each developmental stage (see Figure 1).</li>
	<li>Discontinue rotation and allow the capsules to sink and aspirate the fixative solution into an appropriate waste container.</li>
	<li>Wash the egg capsules by replacing the fixative solution with Phosphate Buffered Saline with 0.1% Tween-20 (PBTw) and rotating at RT for 5 min. Aspirate the PBTw and repeat twice.</li>
	<li>Remove embryos and larvae from their capsules using the apparatus (see section 1 for details) shown in Figure 2. Draw the capsules up into the 20 ml syringe, attach the tubing and then dispel the capsules through the tubing and past the glass needle out into the collection dish. 
	NOTE: Normally, the majority (&#62;90%) of all capsules should need only one pass through the device. In many cases the capsule membrane is damaged but embryos and larvae remain inside the ruptured capsule. Reprocess this material by simply drawing it up into the syringe and dispelling it again.</li>
	<li>Collect the decapsulated embryos and larvae into a 1.5 ml tube using a micropipette (a P20 for 0 - 3 day old larvae, and a P200 for older larvae with the end of the tip cut off). 
	NOTE: A pause (for up to several months) in the protocol can be made at this stage. If this is required, the decapsuled material should be collected into a 1.5 ml tube for storage (continue to the next step). Otherwise continue immediately to Protocol 3.</li>
	<li>Allow embryos and larvae to sink to the bottom of the tube and aspirate the supernatant. Replace with 33% Ethanol (EtOH) in PBTw and let sit for 5 - 10 min. Repeat with 66% EtOH in PBTw and 100% EtOH. Wash larvae twice in 100% EtOH at RT. Store the material at -20 &#176;C in 100% EtOH.</li>
	<li>When ready to continue, re-hydrate the samples by removing the 100% EtOH and replacing with 66% EtOH in PBTw, let sit for 5 - 10 min. Repeat with 33% EtOH in PBTw, let sit for 5 - 10 min. Finally wash with 3x 5 min washes of PBTw to ensure all EtOH is removed.</li>
</ol>
3. Proteinase-K, TEA and Post-fixation
&#160;NOTE: We find performing the following steps in small baskets with a mesh floor the most efficient and gentle method for quickly exchanging time critical solutions. While these can be home made, we use baskets (medium size) that are compatible with the Intavis InSituPro-Vsi liquid handling robot (www.intavis.de/products/automated-ish-and-ihc). Such baskets can be quickly and easily moved between the wells of a 12 well tissue culture dish (TCD) in order to exchange solutions, or the solution can be aspirated from the well using a pipette. Alternatively, all solution exchanges can be performed without these baskets by simply aspirating the supernatant from the larvae. In this case a gentle swirling motion will concentrate all embryos and larvae to the center of the dish allowing the supernatant to be removed from the edge of the well. The following assumes the user is employing baskets for solution exchanges.
<ol>
	<li>Using a pipette, transfer embryos and larvae into a basket sitting in a 12 well TCD with 2 ml of PBTw. 
	NOTE: The number of embryos and/or larvae that can be added depends on the developmental stage being investigated, however a general rule of thumb is to maintain at least 25% of the floor space free of embryos/larvae (i.e., do not overcrowd the basket).</li>
	<li>Prepare an adjacent well with 2 ml of the appropriate Proteinase-K solution (see Figure 1), and another 2 wells with 2 ml of 0.2% Glycine each. Move each basket into the well containing the appropriate concentration of Proteinase-K and immediately start timing.</li>
	<li>After 10 min move each basket into a well containing 0.2% Glycine and incubate for 5 min. Then move each basket into the second well with 0.2% Glycine and incubate for 5 min. Wash out the Glycine with 3x 5 min exchanges of 3 ml PBTw.</li>
	<li>Remove the PBTw solution and treat the samples once with freshly prepared Triethanolamine (TEA) solution for 5 min CAUTION! Do not agitate. Replace with 3 ml of freshly prepared TEA solution for 5 min. Do not agitate. Aspirate the majority of the TEA solution from the samples. Add the freshly prepared Triethanolamine with Acetic Anhydride (TEAAA) solution and incubate for 5 min. CAUTION! Do not agitate.</li>
	<li>OPTIONAL - While the above step is incubating, prepare another fresh batch of TEAAA solution and repeat the above step. 
	NOTE: This second treatment with TEAAA is optional but may help to completely eliminate all background with probes prone to generating background.</li>
	<li>Remove the TEAAA solution by aspirating it from the well and replace with 3 ml of PBTw. Do not agitate. Remove the PBTw and apply 3 ml of 3.7%&#160;formaldehyde in PBTw. Gently swirl occasionally during a 30 min&#160;incubation.</li>
	<li>Remove the fixative by aspirating it from the well and replace with 3 ml of PBTw. Transfer the material into a 1.5 ml tube. Replace the PBTw with hybridization buffer and incubate for 5 min at RT - CAUTION.</li>
	<li>Place the tubes into a hot block at RT, and set the temperature to the desired hybridization temperature. 
	NOTE: The hybridization temperature is probe specific and will need to be empirically optimized, however we find 55 &#176;C to be a good temperature to initially trial. Allow the block to come to the hybridization temperature, and allow the samples to pre-hybridize for approximately 15 min (i.e., the time it takes to prepare the riboprobes, longer is not necessary). Prepare adequate volumes (normally 100 &#181;l) of the diluted riboprobes. We typically trial probe concentrations of 100 and 500 ng/ml as an initial range. We prepare riboprobes according to 12.</li>
	<li>Remove the hybridization buffer from the samples and add the probe in hybridization buffer to the samples. Overlay with 100 &#181;l (or an adequate volume to form a phase above the hybridization buffer) of mineral oil. 
	NOTE: The mineral oil prevents extensive condensation that will form during the lengthy hybridization step. Extensive condensation will significantly alter both the chemistry of the hybridization solution and the concentration of the probe.</li>
	<li>Denature the probe and the target RNA by heating the samples to 75 &#176;C for 10 min, then reduce the heat to the desired hybridization temperature. Allow hybridization to proceed for a minimum of 12 hr (O/N) or longer (24 - 48 hr).</li>
	<li>During hybridization remove a single tube from the heat block, rotate it rapidly between thumb and forefinger to suspend the larvae without disturbing the oil phase, and replace it in the heat block. Repeat this once every 6 - 12 hr or so.</li>
</ol>
4. Hot Washes and Immunodetection
NOTE: While we use a liquid handling robot for the following steps, these can also easily be done manually. In this case, embryos and larvae should be kept in the 1.5 ml tubes they were hybridized in. All subsequent solution exchanges are aspirated and added with a P1,000 pipette. When performed manually each of the following steps should employ 1 ml of each solution.
<ol>
	<li>Heat adequate volumes (3 ml each for each sample) of the 4x, 2x and 1x wash solutions to the hybridization temperature.</li>
	<li>Wash all samples three times in 4x wash buffer for 15 min each at the hybridization temperature. Wash all samples three times in 2x wash buffer for 15 min each at the hybridization temperature. Wash all samples three times in 1x wash buffer for 15 min each at the hybridization temperature.</li>
	<li>Wash all samples once with 1x Sodium Chloride Sodium Citrate buffer + 0.1% Tween (SSC + 0.1% Tween) at the hybridization temperature. Allow samples to cool to RT.</li>
	<li>Wash all samples twice in 1x SSC + 0.1% Tween for 15 min Replace this 1x SSC solution with Maleic Acid Buffer (MAB) and let sit for 10 min Repeat the MAB wash. Replace MAB with block solution and incubate for 1.5 hr.</li>
	<li>Exchange the block solution for antibody solution (1:10,000 dilution of antibody in block solution) and incubate for 12 hr (O/N) at RT with gentle agitation.</li>
</ol>
5. Color Development and Mounting
<ol>
	<li>Aspirate the antibody solution and wash 15 times with PBTw for 10 min each. Replace PBTw with 1x Alkaline Phosphatase Buffer (AP) and incubate for 10 min.</li>
	<li>While the above incubation step proceeds, prepare the AP staining solution (see Supplementary File 2) - CAUTION. Transfer the material into a TCD well and replace the 1x AP solution with AP staining solution. Note the time now so that the length of time the color reaction takes place for can be recorded. Monitor the development of the staining pattern until the signal to background ratio is optimal.</li>
	<li>To stop color development, remove the 1x AP staining solution (dispose in the appropriate waste container), and apply 2 ml of PBTw and note the time now. Rinse twice more with PBTw for 5 min each. Use one of these rinses to transfer the material into a 1.5 ml tube.</li>
	<li>Remove the PBTw and apply 1 ml of 3.7% formaldehyde in PBTw and agitate or rotate for least 30 min at RT (this can also be done O/N).</li>
	<li>Wash out the fixative with 3 washes of PBT (dispose the waste fixative in an appropriate waste container). Wash the samples 3 times at 50 &#176;C in de-ionized water. 
	NOTE: These washes eliminate the precipitation of salts that may become visible during clearing and visualization steps.</li>
	<li>Decide whether the samples should be mounted in Benzyl Benzoate:Benzyl Alcohol (BB:BA, also known as Murray&#39;s clear) or glycerol. 
	NOTE: We prefer the more powerful clearing agent BB:BA as L. stagnalis embryos are somewhat opaque. Samples to be mounted in BB:BA will first need to be dehydrated through an ethanol series. Samples to be mounted in 60% glycerol can be immediately mounted.</li>
	<li>Transfer the samples into the mounting solution (BB:BA or 60% glycerol) using a pipette. 
	NOTE: When mounting in BB:BA this must be done in a glass well (BB:BA will melt polycarbonate plastic).</li>
	<li>Allow the samples to clear for 5 - 10 min, and then mount them onto a slide using an appropriate number of stacked coverslips as spacers (1 coverslip for samples &#60;1 dpfc, 2 coverslips for samples &#62;1 dpfc). 
	NOTE: Whole mounts can now be imaged using a compound microscope with DIC optics.</li>
</ol>

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E., Sarashina, I., Kudoh, T., 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=Left-right+asymmetric+expression+of+dpp+in+the+mantle+of+gastropods+correlates+with+asymmetric+shell+coiling.">Left-right asymmetric expression of dpp in the mantle of gastropods correlates with asymmetric shell coiling.</a> EvoDevo. 4 (1), 15 (2013).</li><li>Christodoulou, F., Raible, F., Tomer, R., Simakov, O., Trachana, K., 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=Ancient+animal+microRNAs+and+the+evolution+of+tissue+identity.">Ancient animal microRNAs and the evolution of tissue identity.</a> Nature. 463, (2010).</li><li>Koga, M., Kudoh, T., Hamada, Y., Watanabe, M., Kageura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+triple+staining+method+for+double+in+situ+hybridization+in+combination+with+cell+lineage+tracing+in+whole-mount+Xenopus+embryos.">A new triple staining method for double in situ hybridization in combination with cell lineage tracing in whole-mount Xenopus embryos.</a> Dev Growth Differ. 49 (8), 635-645 (2007).</li><li>Lauter, G., S&#246;ll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+fluorescent+in+situ+hybridization+in+the+embryonic+zebrafish+brain+using+differential+detection+systems.">Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.</a> BMC Dev. Biol. 11 (1), 43 (2011).</li><li>Davison, A., Frend, H. T., Moray, C., Wheatley, H., Searle, L. J., Eichhorn, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mating+behaviour+in+Lymnaea+stagnalis.+pond+snails+is+a+maternally+inherited,+lateralized+trait.">Mating behaviour in Lymnaea stagnalis. pond snails is a maternally inherited, lateralized trait.</a> Biol. Lett. 5 (1), 20-22 (2009).</li><li>Kuroda, R., Endo, B., Abe, M., Shimizu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+blastomere+arrangement+dictates+zygotic+left-right+asymmetry+pathway+in+snails.">Chiral blastomere arrangement dictates zygotic left-right asymmetry pathway in snails.</a> Nature. 462 (7274), 790-794 (2009).</li><li>Shibazaki, Y., Shimizu, M., Kuroda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+handedness+is+directed+by+genetically+determined+cytoskeletal+dynamics+in+the+early+embryo.">Body handedness is directed by genetically determined cytoskeletal dynamics in the early embryo.</a> Curr. Biol. 14 (16), 1462-1467 (2004).</li><li>Lu, T. Z., Feng, Z. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sodium+leak+current+regulates+pacemaker+activity+of+adult+central+pattern+generator+neurons+in+Lymnaea+stagnalis.">A sodium leak current regulates pacemaker activity of adult central pattern generator neurons in Lymnaea stagnalis.</a> PLoS One. 6 (4), e18745 (2011).</li><li>Dawson, T. F., Boone, A. N., Senatore, A., Piticaru, J., Thiyagalingam, S., 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=Gene+Splicing+of+an+Invertebrate+Beta+Subunit+(LCav-beta)+in+the+N-Terminal+and+HOOK+Domains+and+Its+Regulation+of+LCav1+and+LCav2+Calcium+Channels.">Gene Splicing of an Invertebrate Beta Subunit (LCav-beta) in the N-Terminal and HOOK Domains and Its Regulation of LCav1 and LCav2 Calcium Channels.</a> PLoS ONE. 9 (4), e92941 (2014).</li><li>Smith, S. A., Wilson, N. G., Goetz, F. E., Feehery, C., Andrade, S. C. S., 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=Resolving+the+evolutionary+relationships+of+molluscs+with+phylogenomic+tools.">Resolving the evolutionary relationships of molluscs with phylogenomic tools.</a> Nature. 480 (7377), 364-367 (2011).</li><li>Gregory, T. R., Nicol, J. A., Tamm, H., Kullman, B., Kullman, K., 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=Eukaryotic+genome+size+databases.">Eukaryotic genome size databases.</a> Nuc. Acids. Res. 35 (Database issue), D332-D338 (2007).</li></ol>

The authors have nothing to disclose.

The method described here allows for the efficient visualization of RNA transcripts with presumably varying expression levels within all developmental stages of Lymnaea stagnalis. To remove embryos and larvae from their capsules we trialed a variety of chemical, osmotic shock and physical treatments reported for other encapsulated-developing model organisms. However, in our hands the method we describe here is the only high-throughput technique that removes the tough capsular membrane without damaging the embryo...

Molluscs are a group of animals that hold the interest of a broad diversity of scientific disciplines. Despite their morphological diversity1, species richness (second only to the Arthropods in terms of species number2) and relevance to a wide range of commercial3, medical4 and scientific issues5-8, there are relatively few molluscan species that can claim to be both well-equipped scientific models and easy to maintain in a laboratory environment. One mollusc that is much used by disciplines such as neurobiology9, ecotoxicology10 and more recently evolutionary biology11,12, is Lymnaea stagnalis, primarily because of its widespread distribution and extreme ease of maintenance. Despite its popularity as a &#39;model&#39; organism and its long history of use by developmental biologists13-19, the range and power of molecular tools available to the L. stagnalis scientific community lies far behind that of more traditional animal models (Drosophila, mouse, sea urchin, nematodes).
Our desire to develop Lymnaea as a molecular model stems from an interest in the molecular mechanisms that guide shell formation. This motivated us to refine a set of techniques that would allow for the efficient, consistent and sensitive visualization of gene expression during Lymnaea&#39;s development. Whole mount in situ hybridization (WMISH) is widely employed for a variety of model organisms and has been in use for more than 40 years 20. In its different guises, ISH can be employed to spatially localize specific loci on chromosomes, rRNA, mRNA and micro-RNAs.
One of the challenges we needed to address prior to refining a WMISH method for L. stagnalis was the issue of gently and efficiently extracting embryos and larvae of varying stages from the egg capsules in which they are deposited. This extraction, or &#39;decapsulation&#39;, needs to be achieved efficiently in order to collect adequate material for a given in situ experiment, while at the same time maintaining morphological and cellular integrity. While other model organisms also undergo encapsulated development, in our hands none of the methods reported for those species could be successfully employed in L. stagnalis.
The overall goals of this method are therefore: to extract L. stagnalis embryos and larvae from their capsules in a high-throughput fashion, to apply pre-hybridization treatments that optimize the WMISH signal, to prepare embryos and larvae with satisfactory WMISHsignals for imaging.

<table><tbody><tr><td>Featherweight forceps</td><td>Ehlert &amp;amp; Partner</td><td>#4181119</td><td></td></tr><tr><td>Silicon tubing</td><td>Glasger&amp;auml;tebau OCHS GmbH</td><td>760070</td><td></td></tr><tr><td>Glass capillaries</td><td>Hilgenberg</td><td>1403547</td><td></td></tr><tr><td>12 well tissue culture dishes</td><td>Carl Roth</td><td>CE55.1</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Carl Roth</td><td>P733.1</td><td>CAUTION - May cause cancer. Toxic by inhalation, in contact with skin and if swallowed. Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed.</td></tr><tr><td>Ethylenediamine tetraacetic acid</td><td>Carl Roth</td><td>CN06.3</td><td>CAUTION - CAUSES EYE IRRITATION. MAY CAUSE RESPIRATORY TRACT AND SKIN IRRITATION. Avoid breathing dust. Avoid contact with eyes, skin and clothing. Use only with adequate ventilation</td></tr><tr><td>Magnesium Chloride</td><td>Carl Roth</td><td>2189.1</td><td></td></tr><tr><td>Tween-20</td><td>Carl Roth</td><td>9127.1</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Sodium Chloride</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>Ficoll type 400</td><td>Carl Roth</td><td>CN90.1</td><td></td></tr><tr><td>polyvinylpyrrolidone K30 (MW 40)</td><td>Carl Roth</td><td>4607.1</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Nuclease freeBovine Serum Albumin</td><td>Carl Roth</td><td>8895.1</td><td></td></tr><tr><td>Salmon sperm</td><td>Carl Roth</td><td>5434.2</td><td></td></tr><tr><td>Heparin</td><td>Carl Roth</td><td>7692.1</td><td>CAUTION - ADVERSE EFFECTS INCLUDE HEMORRHAGE, LOCAL IRRITATION. POSSIBLE ALLERGIC REACTION IF INHALED, INGESTED/CONTACTED. EYES/SKIN/RESPIRATORY TRACT IRRITANT. POSSIBLE HYPERSENSITIZATION. DURING PREGNANCY HAS BEEN REPORTED TO INCREASE RISK OF STILLBIRTH</td></tr><tr><td>Proteinase-K</td><td>Carl Roth</td><td>7528.1</td><td></td></tr><tr><td>Glycine</td><td>Carl Roth</td><td>3790.2</td><td></td></tr><tr><td>Deionised formamide</td><td>Carl Roth</td><td>P040.1</td><td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td></tr><tr><td>Standard formamide</td><td>Carl Roth</td><td>6749.3</td><td>CAUTION - Irritating to eyes and skin. May be harmful by inhalation, in contact with skin and if swallowed. May cause harm to the unborn child. Hygroscopic.</td></tr><tr><td>Triethanolamine</td><td>Carl Roth</td><td>6300.1</td><td>CAUTION - Avoid breathing vapor or mist. Avoid contact with eyes. Avoid prolonged or repeated contact with skin. Wash thoroughly after handling.</td></tr><tr><td>Acetic anhydride</td><td>Carl Roth</td><td>4483.1</td><td>CAUTION - CAUSES SEVERE SKIN AND EYE BURNS. REACTS VIOLENTLY WITH WATER. HARMFUL IF SWALLOWED. VAPOR IRRITATING TO THE EYES AND RESPIRATORY TRACT</td></tr><tr><td>Maleic acid</td><td>Carl Roth</td><td>K304.2</td><td>CAUTION - Very hazardous in case of eye contact (irritant), of ingestion, . Hazardous in case of skin contact (irritant), of inhalation (lung irritant). Slightly hazardous in case of skin contact (permeator). Corrosive to eyes and skin.</td></tr><tr><td>Benzyl benzoate</td><td>Sigma</td><td>B6630-250ML</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. Harmful if swallowed.</td></tr><tr><td>Benzyl alcohol</td><td>Sigma</td><td>10,800-6</td><td>CAUTION - Harmful if swallowed. Harmful if inhaled. Causes serious eye irritation.</td></tr><tr><td>Glycerol</td><td>Carl Roth</td><td>3783.1</td><td></td></tr><tr><td>Blocking powder</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>Anti DIG Fab fragments AP conjugated</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>Tris-HCl</td><td>Carl Roth</td><td>9090.3</td><td></td></tr><tr><td>4-Nitro blue tetrazolium chloride in dimethylformamide&amp;nbsp;</td><td>Carl Roth</td><td>4421.3</td><td>CAUTION - May cause harm to the unborn child. Harmful by inhalation and in contact with skin. Irritating to eyes.</td></tr><tr><td>5-bromo-4-chloro-3-indolyl-phosphate</td><td>Carl Roth</td><td>A155.3</td><td>CAUTION - Potentially harmful if ingested. Do not get on skin, in eyes, or on clothing. Potential skin and eye irritant.&amp;nbsp;</td></tr><tr><td>N-acetyl cysteine</td><td>Carl Roth</td><td>4126.1</td><td></td></tr><tr><td>Dithiothreitol</td><td>Carl Roth</td><td>6908.1</td><td>CAUTION - May cause eye and skin irritation. May cause respiratory and digestive tract irritation. The toxicological properties of this material have not been fully investigated.</td></tr><tr><td>Tergitol</td><td>Sigma</td><td>NP40S</td><td>CAUTION - May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed.</td></tr><tr><td>Sodium dodecyl sulphate</td><td>Carl Roth</td><td>CN30.3</td><td>CAUTION - Harmful if swallowed. Toxic in contact with skin. Causes skin irritation. Causes serious eye damage. May cause respiratory irritation.</td></tr><tr><td>Potassium Chloride</td><td>Carl Roth</td><td>6781.1</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Carl Roth</td><td>4984.1</td><td></td></tr><tr><td>Potassium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth</td><td>3904.1</td><td></td></tr><tr><td>Tri sodium citrate dihydrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;Na&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;&amp;#8226;2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Carl Roth</td><td>3580.1</td><td>CAUTION - May cause eye, skin, and respiratory tract irritation. The toxicological properties of this material have not been fully investigated.</td></tr><tr><td>Mineral oil&amp;nbsp;</td><td>Carl Roth</td><td>HP50.2</td><td></td></tr><tr><td>InSituPro-Vsi&amp;nbsp;</td><td>Intavis</td><td></td><td>www.intavis.de/products/automated-ish-and-ihc</td></tr></tbody></table>

a whole mount in situ hybridization method for the gastropod mollusc lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

The representative WMISH staining patterns shown in Figure 3 were generated using the technique described above, and reflect a variety of spatial expression patterns for genes involved in a range of molecular processes ranging from shell formation (Novel gene 1, 2, 3 and 4), to cell-cell signaling (Dpp) to transcription regulation (Brachyury) across a range of developmental stages. While we have not quantified the expression levels of t...

Watch this Scientific Journal Video about A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis at JoVE.com

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

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Research

JoVE Journal

Biology

8.7K Views.  Georg-August University of Göttingen. The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Video: A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

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 ...

Environment

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.

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.

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 ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Developmental Biology

Spatial High-resolution Analysis of Gene Expression Levels in Tendons

In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich tissues, and specifically, tendons were left behind due to their low cell number, low RNA amount per cell, and high matrix content, which made them complicated to analyze. One of the recent and most important single-cell tools is the spatial analysis of gene expression levels in tendons. These RNA spatial tools have specifically high importance in tendons to locate specific cells of new and unknown populations, validate single-cell RNA-seq results, and add histological context to the single-cell RNA-seq data. These new methods will enable the analysis of RNA in cells with exceptional sensitivity and the detection of single-molecule RNA targets at the single-cell level, which will help to molecularly characterize tendons and promote tendon research. 
In this method paper, we will focus on the available methods to analyze spatial gene expression levels on histological sections by using novel in situ hybridization assays to detect target RNA within intact cells at single-cell levels. First, we will focus on how to prepare the tendon tissue for the different available assays and how to amplify target-specific signals without background noise but with high sensitivity and high specificity. Then, the paper will describe specific permeabilization methods, the different probe designs, and the signal amplification strategies currently available. These unique methods of analyzing transcription levels of different genes in single-cell resolution will enable the identification and characterization of the tendon tissue cells in young and aged populations of various animal models and human tendon tissues. This method will also help analyze gene expression levels in other matrix-rich tissues such as bones, cartilage, and ligaments.

This article describes how to perform an optimized in situ protocol for tendons. This method discusses tissue preparation, section permeabilization, probe design, and signal amplification methods.

In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich ...

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 ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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 ...

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

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. ...

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Summary

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A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

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

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In situ Hybridization for Sipunculus nudus Coelomic Fluid

Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection