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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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><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&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</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&amp;nbsp;</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 &amp;micro;L tip</td></tr><tr><td>Hairpins&amp;nbsp;</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&amp;nbsp;</td><td>AGAP010272</td></tr><tr><td>IR41t.1 probe</td><td>Molecular Instruments</td><td>&amp;nbsp;Probe Set ID: PRK978</td><td>AGAP004432</td></tr><tr><td>IR64a probe</td><td>Molecular Instruments</td><td>Probe Set ID: PRK700&amp;nbsp;</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 (MgCl&lt;sub&gt;2&lt;/sub&gt;) 1M</td><td>Thermo Fisher</td><td>AM9530G</td><td></td></tr><tr><td>Methanol</td><td>Fisher&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Thermo Fisher</td><td>15-557-044</td><td></td></tr><tr><td>SlowFade Diamond</td><td>Thermo Fisher&amp;nbsp;</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&amp;nbsp; (10%)</td><td>Sigma-Aldrich&amp;nbsp;</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>&amp;nbsp;1 5/8&quot; square; transparent</td></tr></tbody></table>

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 (...

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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 ...

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Research

JoVE Journal

Neuroscience

1.2K 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 ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

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 ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Biology

Fluorescence In Situ Hybridization to Study Mosquito Spermatogenesis

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Author Spotlight: Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

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

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Double Fluorescence in situ Hybridization in Fresh Brain Sections

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

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

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

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti