We thank Margo Herre and the Leslie Vosshall lab for sharing their in-situ hybridization protocol for Aedes aegypti olfactory appendages. This work was supported by grants from the National Institutes of Health to C.J.P. (NIAID R01Al137078), a HHMI Hanna Gray fellowship to J.I.R, a Johns Hopkins Postdoctoral Accelerator Award to J.I.R, and a Johns Hopkins Malaria Research Institute Postdoctoral Fellowship to J.I.R. We thank the Johns Hopkins Malaria Research Institute and Bloomberg Philanthropies for their support.

1. Considerations and preparation of materials
<ol>
	<li>Decide if whole-mount or cryo-section of tissue will be appropriate. This protocol is optimized for whole-mount in situ imaging of RNA in the Anopheles mosquito antenna and maxillary palp without cryo-sectioning. If samples are thicker than 5 mm, cryo-sectioning is recommended to enable probe penetration.</li>
	<li>Identify the genes of interest and copy the sequences including introns and exons from a suitable database. Transcribe the gene sequence into RNA for synthesis.</li>
	<li>Determine whether to purchase probes from commercial vendors or if probes will be synthesized in the laboratory. 
	NOTE: In this study, the in situ probes were purchased from a commercial vendor (see Table of Materials). Alternatively, it can be synthesized as previously reported10. Reagents used in the study are described in Table 1. Materials required to prepare the reagents are listed in the Table of Materials.</li>
</ol>
2. Tissue pre-fixation
<ol>
	<li>Anesthetize 10-15 adult female or male Anopheles coluzzii mosquitoes (strain N&#39;Gousso), aged 5-10 days post emergence, by collecting them with a mouth aspirator into a paper cup or plastic tube and placing them in a bucket containing ice. A successful cold-induced anesthesia can be confirmed when the mosquitoes are immobile.</li>
	<li>Decapitate them by removing the heads from the neck region with a pair of forceps. Using the non-dominant hand grab the thorax with forceps and separate the neck from the body with forceps held by the dominant hand. Place heads in a 1.5 mL tube containing 500 &#181;L of chitinase-chymotrypsin dimethyl sulfoxide buffer (CCD buffer) on ice. 
	NOTE: Freezing animals can deform the antennae; a quick knockdown on ice is recommended. The mosquito&#39;s age during testing may vary depending on the project. It is important to confirm and coordinate their physiological status, such as whether they are blood-fed, starved, or mated. In this study, olfactory appendages were sampled from mosquitoes that had been blood-fed and mated.</li>
	<li>Pre-heat mosquito heads in CCD buffer at 37 &#730; C on a heat block for 5 min and then transfer the tube containing the mosquito heads to a hybridization oven and rotate at 37 &#730; C. Incubation time in CCD buffer depends on tissue type. For female Anopheles antennae, use 20 min, male antennae require 15 min, while longer incubation time (1 h) is needed for the maxillary palps.</li>
	<li>Transfer the entire content of the tube into a dissecting watch glass. Gently pour sample into the depression of a watch (dissection) glass. If mosquito heads are stuck inside the tube, use a pipette to add CCD buffer into the tube and rinse out the head.</li>
	<li>Use forceps to carefully transfer the heads or any antennae/palps detached during incubation and fix in 1 mL of the pre-fixative. 
	NOTE: Leftover CCD buffer can be preserved at -20 &#730; C.&#160;Buffer may be reused up to 3x. If the pre-fixative turns slightly brownish because of the CCD buffer carryover by the tissues, replace it with another 1 mL of fixative.</li>
	<li>Rotate heads in pre-fixative for 24 h at 4&#730; C using a nutator. 
	&#8203;NOTE: Rocking speed for the nutator used in this study was non-adjustable; the default speed set (12 rpm) by the manufacturer was used.</li>
</ol>
3. Tissue dissection
<ol>
	<li>Rinse heads 4x (5 min per wash) with 1 mL of 0.1% PBS-Tween on ice. To avoid sample loss, use a pipette attached to gel loading tip to remove the liquid. 
	NOTE: Two quick washes followed by a 10 min long wash, and a final quick wash could be performed instead of step 3.1.</li>
	<li>Transfer the heads in the tube into a dissecting watch glass. Gently pour sample into the depression of a watch glass. Rinse out mosquito heads that are stuck inside the tube with 0.1% PBS-Tween.</li>
	<li>Under a dissecting microscope, remove tissues of interest (antennae/palps) from the head with sharp forceps. Hold the posterior part of the head with forceps and grab an antenna with another forceps from the base. Clean forceps with paper moistened with RNAse-free solvent. Remove the palps using the same process.</li>
	<li>Transfer the antennae and palps with forceps into empty DNA/RNase-free tubes placed on ice. Separate the different tissue parts into pre-labeled different tubes.</li>
	<li>Dehydrate tissue in 400 &#181;L of solvent containing a mixture of methanol (MeOH 80%) and Dimethyl sulfoxide (DMSO 20%) for 1 h at room temperature. 
	NOTE: There is no need to place tissue on a nutator; let it sit on a tube rack at room temperature on the lab bench. It is recommended to use fresh solvent mixture. For a 500 &#181;L stock solution, mix 400 &#181;L of MeOH with 100 &#181;L of DMSO.</li>
	<li>Replace the dehydrating reagent with 400 &#181;L of absolute (100%) methanol and dehydrate tissues overnight at -20 &#730;C. Allow tissues to settle by gravity and use a pipette to remove the liquid. 
	&#8203;NOTE: Samples are viable for extended period of dehydration, up to 4 nights have been tested without loss of signal.</li>
</ol>
4. Tissue post-fixation
<ol>
	<li>Rehydrate tissues in a four-step series of graded 400 &#181;L MeOH/PBS-Tween for 10 min on ice. Start with 75% MeOH/25% PBS-Tween followed by 50% MeOH/ 50% PBS-Tween, then 25% MeOH/ 75% PBS-Tween and finally 100% PBS-Tween. 
	NOTE: A gel loading tip with a tiny opening can be used to remove the buffer from the tube to avoid losing the tissues. Buffer removal can be done under a dissecting microscope.</li>
	<li>Wash with 400 &#181;L of phosphate-buffered saline containing 0.1% Tween-20 (PBS-T) for 10 min at room temperature. Wash by placing the sample on a nutator.</li>
	<li>Make a 20 &#181;g/mL Proteinase-K solution and incubate in 400 &#181;L of Proteinase-K solution for 30 min at room temperature. 
	NOTE: Dilute 20 mg/mL Proteinase-K stock (1000x stock solution) i.e., 2 &#181;L in 2 mL of 0.1% PBS-Tween. The left over can be stored in a -20 &#730; C freezer.</li>
	<li>Stop enzymatic digestion of tissue by washing 2x for 10 min with 400 &#181;L of 0.1% PBS-tween.</li>
	<li>Add 400 &#181;L of the post-fixative and incubate for 20 min at room temperature. Wash 3x, 15 min per wash, with 400 &#181;L of 0.1% PBS-Tween.</li>
</ol>
5. Probe hybridization
<ol>
	<li>Incubate tissue in 400 &#181;L of probe hybridization buffer for 5 min. Ensure tissue is completely submerged in the buffer by gently pipetting.</li>
	<li>Prepare for the next step by heating an aliquot of probe hybridization buffer to 37 &#730; C for 30 min.</li>
	<li>Remove buffer and pre-hybridize with 400 &#181;L of pre-heated probe hybridization buffer for 30 min at 37 &#730; C.</li>
	<li>Make probe solution for the target chemosensory receptors (IR8a, IR76b, IR25a, IR41t.1, IR75d, IR7t, IR64a or Orco) by adding 8 pM of probe. Add 8 &#181;L of 1 &#181;M probe stock to 500 &#181;L pre-heated probe hybridization buffer.</li>
	<li>Remove the pre-heated probe hybridization buffer and replace it with 400 &#181;L of heated probe solution.</li>
	<li>Incubate tissue at 37 &#730; C for two nights on a nutator placed inside an incubator and covered under a box.</li>
</ol>
6. Probe amplification
<ol>
	<li>Heat up the probe wash buffer to 37 &#730; C. Remove excess probe solution by rinsing the tissue 5x, 10 min per wash, with 400 &#181;L of probe wash buffer at 37 &#730; C, nutating in the incubator. 
	NOTE: Preparation for step 6.4 may begin. See the note in step 6.4.</li>
	<li>Wash samples 2x, 5 min per wash, with 400 &#181;L of 5x saline-sodium citrate containing 10% Tween-20 (SSCT) at room temperature. Thaw amplification buffer to room temperature on a bench top.</li>
	<li>Prepare tissue for amplification by incubating with 400 &#181;L of amplification buffer for 10 min at room temperature. Due to the viscosity of the amplification buffer, tissues may not be completely submerged. Mix by gently pipetting the amplification buffer below the tissue and expelling the buffer on top of the tissue until they are submerged.</li>
	<li>Separately prepare 18 pM of hairpin h1 and 18 pM of hairpin h2 by heating 6 &#181;L of 3 &#181;M stock at 95 &#176;C for 90 s and cool to room temperature in a dark drawer for 30 min. Ensure PCR tubes are tightly capped to prevent evaporation of the hairpins while heating in a thermal cycler. 
	NOTE: To save time, step 6.4 can be initiated while on the 4th washing step in step 6.1. Hairpins should be separately heated to prevent cross reaction. Hairpins should not be diluted with any buffer in this step. The hairpins can be purchased from the probe manufacturer along with the probes.</li>
	<li>Replace the amplification buffer added in step 6.3 with a mixture containing the heated hairpins (from step 6.4) together with 100 &#181;L of amplification buffer. A typical reaction will contain 6 &#181;L of h1, 6 &#181;L of h2, and 100 &#181;L of amplification buffer. 
	NOTE: Hairpins h1 and h2 can be combined after cooling to room temperature and then added to 100 &#181;L of amplification buffer. To avoid transferring the tissue samples from one tube to another, the amplification buffer in step 6.3 can be removed and replaced by the hairpins mixture diluted in amplification buffer.</li>
	<li>Incubate tissue overnight and nutate in the dark at room temperature using the default speed of the nutator.</li>
</ol>
7. Mounting tissue sample
<ol>
	<li>Dilute amplification buffer in the incubated tissue with 300 &#181;L of 5x SSCT (sodium chloride-sodium citrate diluted in Tween-20). 
	NOTE: The dilution is helpful to reduce the viscosity and makes it easier to remove the amplification buffer from the tissue. We used Triton-X 100 for the pre-fixative and Tween-20 for the post-fixative step because of the differences in their actions as agents to permeabilize cell membrane.</li>
	<li>Wash tissue 5x with 400 &#181;L of 5x SSCT at room temperature. 
	NOTE: Store tissue temporarily at 4 &#176;C until ready to be mounted, if needed. We have not exceeded 2 days before imaging.</li>
	<li>Make 5 droplets of mounting solution on a glass slide. Cut the tip of a 200 &#181;L pipette tip to make it wider and transfer the tissue to a new glass slide.</li>
	<li>Grab the tissue samples by their base with forceps and gently immerse and rinse in a series of mounting solution droplets. Be careful not to break the tissues in this step.</li>
	<li>Mount with mounting solution, place coverslip, and seal with nail polish. Image in situ tissue samples using a confocal microscope.</li>
</ol>

Chemosensory Gene Expression Mapping in Mosquito Olfactory Appendages

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	<li>Konopka, J. 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=Olfaction+in+Anopheles+mosquitoes.">Olfaction in Anopheles mosquitoes.</a> Chem Senses. 46, (2021).</li><li>Raji, J. I., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+ionotropic+receptors+in+human+host-seeking+mosquitoes.">Chemosensory ionotropic receptors in human host-seeking mosquitoes.</a> Curr Opin Insect Sci. 54, 100967 (2022).</li><li>Young, A. P., Jackson, D. J., Wyeth, R. 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+technical+review+and+guide+to+RNA+fluorescence+in+situ+hybridization.">A technical review and guide to RNA fluorescence in situ hybridization.</a> PeerJ. 8, e8806 (2020).</li><li>Herre, M., 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=Non-canonical+odor+coding+in+the+mosquito.">Non-canonical odor coding in the mosquito.</a> Cell. 185 (17), 3104-3123.e28 (2022).</li><li>Raji, J. I., Konopka, J. K., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+map+of+antennal-expressed+ionotropic+receptors+in+the+malaria+mosquito.">A spatial map of antennal-expressed ionotropic receptors in the malaria mosquito.</a> Cell Rep. 42 (2), 112101 (2023).</li><li>Choi, H. M. 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=Programmable+in+situ+amplification+for+multiplexed+imaging+of+mRNA+expression.">Programmable in situ amplification for multiplexed imaging of mRNA expression.</a> Nat Biotechnol. 28 (11), 1208-1212 (2010).</li><li>Choi, H. M. T., Beck, V. A., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+in+situ+hybridization+chain+reaction:+higher+gain,+lower+cost,+greater+durability.">Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.</a> ACS Nano. 8 (5), 4284-4294 (2014).</li><li>Choi, H. M. 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=Third-generation+in+situ+hybridization+chain+reaction:+multiplexed,+quantitative,+sensitive,+versatile,+robust.">Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</a> Development. 145 (12), dev165753 (2018).</li><li>Schwarzkopf, M., 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=Hybridization+chain+reaction+enables+a+unified+approach+to+multiplexed,+quantitative,+high-resolution+immunohistochemistry+and+in+situ+hybridization.">Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization.</a> Development. 148 (22), dev199847 (2021).</li><li>Choi, H. M. 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=Mapping+a+multiplexed+zoo+of+mRNA+expression.">Mapping a multiplexed zoo of mRNA expression.</a> Development. 143 (19), 3632-3637 (2016).</li><li>Pitts, R. J., Derryberry, S. L., Zhang, Z., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+ionotropic+receptors+in+the+malaria+vector+mosquito+Anopheles+gambiae+tuned+to+amines+and+carboxylic+acids.">Variant ionotropic receptors in the malaria vector mosquito Anopheles gambiae tuned to amines and carboxylic acids.</a> Sci Rep. 7, 40297 (2017).</li><li>Rinker, D. C., Zhou, X., Pitts, R. J., Rokas, A., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+transcriptome+profiles+of+anopheline+mosquitoes+reveal+human+host+olfactory+specialization+in+Anopheles+gambiae.">Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae.</a> BMC Genomics. 14, 749 (2013).</li><li>Maguire, S. E., Afify, A., Goff, L. A., Potter, C. 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The authors have nothing to disclose.

The third generation of hybridization chain reaction (HCR) is remarkable for its sensitivity and robustness to visualize several RNA targets8. HCR WM-FISH has been successfully used on the embryos of Drosophila, chicken, mice, and zebrafish as well as the larvae of nematodes and zebrafish10,16,17. Mosquito antennae and maxillary palps are typically prone to high autofluorescence and weak probe pe...

Mosquito vectors such as Anopheles gambiae rely on a rich repertoire of chemosensory genes expressed in their peripheral olfactory appendages to thrive in a complex chemical world and identify behaviorally relevant odors emanating from human hosts, detect nectar sources, and locate oviposition sites1. The mosquito antenna and the maxillary palp are enriched with chemosensory genes that drive odor detection in these olfactory appendages. Three main classes of ligand-gated ion channels drive odor detection in mosquitoes&#39; olfactory appendages: the Odorant receptors (ORs), which function with an obligate Odorant receptor co-receptor (Orco); the Ionotropic receptors (IRs), which interact with one or more IR coreceptors (IR8a, IR25a, and IR76b); the chemosensory Gustatory receptors (GRs), which function as a complex of three proteins to detect carbon dioxide (CO2)1,2.
RNA fluorescence in situ hybridization is a powerful tool for detecting the expression of endogenous mRNA3. In general, this method utilizes a fluorophore-tagged single stranded nucleic acid probe with sequence complementary to a target mRNA. Binding of the fluorescent RNA probe to the target RNA allows identification of cells expressing a transcript of interest. Recent advancements now enable the detection of transcripts in whole-mount mosquito tissues4,5. The first generation of hybridization chain reaction (HCR) technology used an RNA-based HCR amplifier; this was improved upon in a second-generation method that instead used engineered DNA for the HCR amplifier6,7. This upgrade resulted in a 10x increase in signal, a dramatic decrease in production cost, and significant improvement in the durability of reagents6,7.
In the protocol, we describe the utilization of a third generation HCR whole-mount RNA fluorescence in situ hybridization (HCR RNA WM-FISH) method designed for detecting the spatial localization and expression of any gene8,9. This two-step method first utilizes nucleic acid probes specific for the mRNA of interest, but which also contain an initiator recognition sequence; the second step utilizes fluorophore-tagged hairpins which bind to the initiator sequence to amplify the fluorescent signal (Figure 1). This method also allows for the multiplexing of two or more RNA probes and amplifying probe signals to facilitate RNA detection and quantification8. Visualizing the transcript abundance and RNA localization patterns of chemosensory genes expressed in the olfactory appendages offers the first line of insight into chemosensory gene functions and odor coding.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amplification buffer</td>
			<td>Molecular Instruments</td>
			<td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2) 1M&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>21115-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Chitinase</td>
			<td>Sigma-Aldrich</td>
			<td>C6137-50UN</td>
			<td></td>
		</tr>
		<tr>
			<td>Chymotrypsin</td>
			<td>Sigma-Aldrich</td>
			<td>CHY5S-10VL&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>VWR</td>
			<td>20901-551</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11251</td>
			<td>Number 5</td>
		</tr>
		<tr>
			<td>Gel loading tip</td>
			<td>Costar</td>
			<td>4853</td>
			<td>1-200 &#181;L tip</td>
		</tr>
		<tr>
			<td>Hairpins&#160;</td>
			<td>Molecular Instruments</td>
			<td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td>
			<td>h1 and h2 initiator splits</td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>IR25a probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK149&#160;</td>
			<td>AGAP010272</td>
		</tr>
		<tr>
			<td>IR41t.1 probe</td>
			<td>Molecular Instruments</td>
			<td>&#160;Probe Set ID: PRK978</td>
			<td>AGAP004432</td>
		</tr>
		<tr>
			<td>IR64a probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK700&#160;</td>
			<td>AGAP004923</td>
		</tr>
		<tr>
			<td>IR75d probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK976</td>
			<td>AGAP004969</td>
		</tr>
		<tr>
			<td>IR76b probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRI998</td>
			<td>AGAP011968</td>
		</tr>
		<tr>
			<td>IR7t probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRL355</td>
			<td>AGAP002763</td>
		</tr>
		<tr>
			<td>IR8a probe</td>
			<td>Molecular Instruments</td>
			<td>Probe Set ID: PRK150</td>
			<td>AGAP010411</td>
		</tr>
		<tr>
			<td>LoBind Tubes</td>
			<td>VWR</td>
			<td>80077-236</td>
			<td>0.5 mL DNA/RNA LoBind Tubes</td>
		</tr>
		<tr>
			<td>Magnessium Chloride (MgCl2) 1M</td>
			<td>Thermo Fisher</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher&#160;</td>
			<td>A412-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Thermo Fisher</td>
			<td>43-879-36</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Denville Scientific</td>
			<td>Model 135</td>
			<td>3-D Mini rocker</td>
		</tr>
		<tr>
			<td>Orco probe</td>
			<td>Molecular Instruments</td>
			<td>Probe set ID PRD954</td>
			<td>AGAP002560</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (20% )</td>
			<td>Electron Microscopy Services&#160;</td>
			<td>15713-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10X PBS)</td>
			<td>Thermo Fisher</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe hybridization buffer</td>
			<td>Molecular Instruments</td>
			<td>https://www.molecularinstruments.com/</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Probe wash buffer</td>
			<td>Molecular Instruments</td>
			<td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Proteinase-K</td>
			<td>Thermo Fisher</td>
			<td>AM2548</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline-Sodium Citrate (SSC) 20x&#160;</td>
			<td>Thermo Fisher</td>
			<td>15-557-044</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFade Diamond</td>
			<td>Thermo Fisher&#160;</td>
			<td>S36972</td>
			<td>mounting solution</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl) 5M</td>
			<td>Invitrogen</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160; (10%)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20 (10% )</td>
			<td>Teknova</td>
			<td>T0027</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass</td>
			<td>Carolina</td>
			<td>742300</td>
			<td>&#160;1 5/8&#34; square; transparent</td>
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hybridization chain reaction rna whole-mount fluorescence in situ hybridization of chemosensory genes in mosquito olfactory appendages

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory appendages. Understanding how chemosensory receptors are spatially organized in the peripheral olfactory appendages can offer insights into how odor is encoded in the mosquito olfactory system and inform new ways to combat the spread of mosquito-borne diseases. The emergence of third-generation hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) allows for spatial mapping and simultaneous expression profiling of multiple chemosensory genes. Here, we describe a stepwise approach for performing HCR RNA WM-FISH on the Anopheles mosquito antenna and maxillary palp. We investigated the sensitivity of this technique by examining the expression profile of ionotropic olfactory receptors. We asked if the HCR WM-FISH technique described was suitable for multiplexed studies by tethering RNA probes to three spectrally distinct fluorophores. Results provided evidence that HCR RNA WM-FISH is robustly sensitive to simultaneously detect multiple chemosensory genes in the antenna and maxillary palp olfactory appendages. Further investigations attest to the suitability of HCR WM-FISH for co-expression profiling of double and triple RNA targets. This technique, when applied with modifications, could be adaptable to localize genes of interest in the olfactory tissues of other insect species or in other appendages.

Robust detection of chemosensory genes in Anopheles antenna 
We investigated the sensitivity of the HCR FISH method (Figure 1) to detect the expression of chemosensory receptors in mosquito olfactory tissues. Guided by the RNA transcript data reported earlier on the female Anopheles mosquito antenna, we generated probes to target a variety of IRs. The average transcript values from four independent antennal transcriptome studies revealed that Ir41t.1 (...

Watch this Scientific Journal Video about Visualizing Olfactory Receptor Expression in Mosquitoes at JoVE.com

Author Spotlight: Visualizing Olfactory Receptor Expression in Mosquitoes 

The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp.

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

hybridization-chain-reaction-rna-whole-mount-fluorescence-situ

Research

JoVE Journal

Neuroscience

1.1K Views.  Johns Hopkins University School of Medicine. The article describes the methods and reagents necessary to perform hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) to reveal insights into the spatial and cellular resolution of chemosensory receptor genes in the mosquito antenna and maxillary palp. 

Video: Author Spotlight: Visualizing Olfactory Receptor Expression in Mosquitoes 

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

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

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

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

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

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

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...