This work is supported by a grant&#160;from National Natural Science Foundation of China (No.31472037). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation.

NOTE: To limit RNA degradation, prepare solutions using wet-autoclaved distilled water (at 121 &#176;C for 60 min) and also wet-autoclave materials.
1. Preparation of RNA ISH Antisense and Sense Probes
<ol>
	<li>Target gene amplification and purification
	<ol>
		<li>First, produce a 387 bp double-stranded fragment of L. migratoria OR1 (LmigOR1, GenBank: JQ766965) from the plasmid containing the full-length cDNA of LmigOR1 with a Taq DNA Polymerase that adds adenines to both ends of the fragments.
		<ol>
			<li>Use a 100 &#181;L final reaction volume, containing 50 &#181;L of 2x reaction mix, 4 &#181;L each of sense and antisense primers (Table 1), 1 &#181;L of plasmid template, and 41 &#181;L of RNase-free H2O. Use the following PCR protocol: 94 &#176;C for 5 min, followed by 30 cycles of 94 &#176;C for 30 s, 55 &#176;C for 30 s and 72 &#176;C for 1 min, followed by 72 &#176;C for 10 min.</li>
			<li>Repeat these steps to produce the 1,303 bp double-stranded fragment of LmigOR2 (GenBank: JQ766966) and 1,251 bp double-stranded fragment of LmigORco (GenBank: JN989549). The primers used in these experiments are presented in Table 1.</li>
			<li>Run PCR products on 1.2% agarose gels in 1x Tris-acetate-EDTA buffer and visualize using ethidium bromide staining.</li>
		</ol>
		</li>
		<li>Extract these PCR products using a gel extraction kit.
		<ol>
			<li>Following electrophoresis, excise DNA bands from the gel and place the gel slices in a 1.5 mL tube. Then, add 100 &#181;L of binding buffer (Buffer PN) per 0.1 g of gel slice. Vortex and incubate at 50 &#176;C until the gel slice is completely dissolved.</li>
			<li>Insert a CA2 adsorption column into the collection tube and add 500 &#181;L of balance buffer (Buffer BL). This improves the absorption capability and stability of the silica membrane. Centrifuge at 12,400 x g for 1 min.</li>
			<li>Transfer the dissolved gel mixture to the adsorption column assembly and incubate&#160;at room temperature for 2 min. Then, centrifuge tubes at 12,400 x g for 1 min. Discard the flow-through and reinsert the adsorption column into collection tube.</li>
			<li>Add 600 &#181;L of wash solution (ethanol added, Buffer PW) twice. Centrifuge at 12,400 x g for 1 min. Discard flow-through and reinsert the adsorption column into collection tube.</li>
			<li>Empty the collection tube and recentrifuge the column assembly at 12,400 x g for 2 min to allow evaporation of any residual ethanol.</li>
			<li>Carefully transfer adsorption column to a clean 1.5 mL centrifuge tube. Air-dry the pellet for 5-10 min and redissolve the DNA in a suitable volume (e.g., 30 &#181;L) of nuclease-free water.</li>
			<li>Incubate at RT for 2 min. Centrifuge at 12,400 x g for 2 min. Discard adsorption column and store the DNA at 4 &#176;C or -20 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Construct the recombinant plasmids
	<ol>
		<li>Individually ligate the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2&#160;and 1,251 bp fragment of LmigORco into a T vector that contains promoters for T7 (upstream) and SP6 (downstream) RNA polymerases adjacent to the inserted DNA using T4 DNA ligase. Prepare the following 10 &#181;L reaction: 5 &#181;L of 2x ligation buffer, 1 &#181;L of T vector, 3 &#181;L (&#8764;100 ng) of the inserted gene&#39;s DNA and 1 &#181;L of T4 DNA ligase, and incubate O/N at 4 &#176;C.</li>
		<li>Add 5 &#181;L of each recombinant plasmid containing the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2&#160;and 1,251 bp fragment of LmigORco separately into 50 &#181;L of competent Escherichia coli DH5&#945; cells in sterile 1.5 mL tubes.
		<ol>
			<li>Mix gently and put the tubes into ice for 30 min and then incubate them for 90 s at 42 &#176;C. Transfer them back into ice for 3 min.</li>
			<li>Add 450 &#181;L of Luria-Bertani (LB) liquid medium without ampicillin to every tube and incubate them in a shaker at 150 rpm for 1 h at 37 &#176;C to restore the E. coli&#160;DH5&#945;.</li>
			<li>Take a 100 &#181;L aliquot of each transformant and use them to inoculate LB solid substrate plates containing 50 &#181;g/mL ampicillin, 24 &#181;g/mL isopropyl &#946;-D-1-thiogalactopyranoside (IPTG), and 40 &#181;g/mL 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-gal).</li>
			<li>Incubate these plates in a constant incubator at 37 &#176;C for 18 h. Screen the target recombinant plasmids using the blue-white screening method. Sequence the target recombinant plasmids using Sanger sequencing and identify the three OR genes&#39; insert directions.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Select restriction endonucleases to linearize recombinant plasmids
	<ol>
		<li>Use restriction endonucleases that not only digest in the multiple cloning site of the vector but also do not cut the insert DNA. In this experiment, all 3 genes are inserted into the T vector in the direction corresponding to that of the vector.</li>
		<li>Use Nco I restriction endonuclease to linearize the recombinant plasmids containing the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2 and 1,251 bp fragment of LmigORco to produce the antisense probes. Use Spe I restriction endonuclease to produce the sense probes. 
		NOTE: If the insert direction corresponds to that of the vector, then a unique restriction site from the end of T7 is selected to linearize the recombinant plasmid and SP6 RNA polymerase is used to prepare the antisense probe. A unique restriction site from the end of SP6 is selected to linearize the recombinant plasmid, and the T7 RNA polymerase is used to prepare the sense probe. Otherwise, if the insert direction does not correspond to the vector&#39;s direction, then a unique restriction site from the end of SP6 is selected to linearize the recombinant plasmid and T7 RNA polymerase is used to prepare the antisense probe. The unique restriction site from the end of T7 is selected to linearize the recombinant plasmid, and the SP6 RNA polymerase is used to prepare sense probe.</li>
	</ol>
	</li>
	<li>Purifying the linearized recombinant plasmids
	<ol>
		<li>Digest 20 &#181;g each of the three recombinant plasmids containing the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2 and 1,251 bp fragment of LmigORco using the Nco I restriction endonuclease in a 100 &#181;L reaction that contains 10 &#181;L of 10x buffer, 40 &#181;L of 0.5 &#181;g/&#181;L plasmid, 3 &#181;L&#160;of Nco I and 47 &#181;L of RNase-free H2O, followed by a 3 h incubation at 37 &#176;C.</li>
		<li>Similarly, digest 20 &#181;g of each of these three recombinant plasmids using the Spe I restriction endonuclease.</li>
		<li>Purify these digested plasmids using a gel extraction kit as described in 1.1.2. Determine the concentrations of these extracted linearized recombinant plasmids by spectrophotometry and adjust to 0.5 &#181;g/&#181;L.</li>
	</ol>
	</li>
	<li>Prepare antisense and sense probes
	<ol>
		<li>Use the T7/SP6 RNA transcription system to generate antisense and sense probes. SP6 RNA polymerase is used to transcribe double-stranded RNA for DIG- and biotin-labeled antisense probes for these three OR genes using linearized recombinant plasmids as templates in vitro. The T7 RNA polymerase is used to produce sense probes. Perform the reaction in a 20 &#181;L final volume, containing 2 &#181;L of 10x NTP mix, 2 &#181;L of 10X transcription buffer, 1 &#181;L of RNase inhibitor, 2 &#181;L of T7 or SP6 RNA polymerase, 4 &#181;L of 0.5 &#181;g/&#181;L linearized plasmid and 9 &#181;L RNase-free H2O, followed by a 3 h incubation at 37 &#176;C.</li>
		<li>Incubate the DIG- and biotin-labeled antisense and sense probes for 30 min with 1 &#181;L DNase at 37 &#176;C. Add 2.5 &#181;L of 5 M LiCl and 75 &#181;L of absolute ethanol, then incubate the reactions for 30 min at -70 &#176;C.</li>
		<li>Centrifuge at 15,000 x g for 30 min at 4 &#176;C. Carefully decant the supernatant. Add 150 &#181;L of 70% ethanol to wash the precipitant. Prepare the 70% ethanol (1 L) by adding 95% ethanol (736.8 mL) to sterile distilled water (263.2 mL).</li>
		<li>Centrifuge at 15,000 x g for 30 min at 4 &#176;C again and air-dry the pellet for 5-10 min at RT. Add 25 &#181;L of RNase-free H2O to dissolve the RNA antisense and sense probes.</li>
		<li>If the length of the inserted gene is longer than 1 kb, subsequently fragment RNA antisense and sense probes to an average length of &#65374;300 bp by incubation in a bicarbonate-carbonate buffer solutions. Perform the reaction in a 50 &#181;L final volume, containing 25 &#181;L of the RNA probe and 25 &#181;L of the bicarbonate-carbonate buffer solution (80 mM NaHCO3, 120 mM Na2CO3, pH 10.2) at 60 &#176;C. The hydrolysis time required is given by a previously published formula17.</li>
		<li>Add 5 &#181;L of 10% acetic acid to stop the reaction. In this experiment, fragment the LmigOR2 and LmigORco antisense and sense probes for 24 and 23 min, respectively. 
		t = (Lo-Lf)/kXLoXLf 
		Lo= the initial fragment lengths in kb 
		Lf=the final fragment lengths in kb 
		k=the rate constant for hydrolysis, approximately 0.11 kb-1min-1 
		t=the hydrolysis time in min</li>
		<li>Finally, add 250 &#181;L of hybridization buffer and store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of Cryostat Sections
<ol>
	<li>Precool the freezing microtome to -24 &#176;C.</li>
	<li>Select new molting adult locusts that are active and have intact antennae. Cut the antennae into 2-3 mm pieces using sterile razors.</li>
	<li>Put O.C.T. compound on a freezing microtome holder and put one or two samples on the compound horizontally. Then, transfer the holder into the freezing microtome at -24 &#176;C to equilibrate until the compound freezes. Take out the holder and cover the samples with a little compound. Transfer the holder into the freezing microtome at -24 &#176;C again for at least 10 min (Figure 1). 
	NOTE: Avoid getting bubbles in the compound.</li>
	<li>Fix the holder with the samples into the freezing microtome, then section the frozen samples into 12 &#181;m-thick slices at -24 &#176;C.</li>
	<li>Thaw mount the slices one by one on slides (25 &#160;x 75 mm; nuclease-free) and air dry for 10 min.</li>
</ol>
3. Fixing Sections
<ol>
	<li>After preparing cryostat sections, put the slides in a plastic container (&#65374;100 &#160;x 40 &#160;x 80 mm), and fix the tissues by incubating the slides in 4% paraformaldehyde solution (PFA) for 30 min at 4 &#176;C.</li>
	<li>Wash the slides in 1X Phosphate-Buffered Saline (PBS) for 1 min.</li>
	<li>To eliminate the alkaline proteins, transfer the slides to 0.2 M HCl for 10 min.</li>
	<li>To eliminate the surface protein of nucleic acid, transfer the slides to 1XPBS with 1% Triton X-100 for 2 min.</li>
	<li>Wash the slides twice for 30 s in 1x PBS.</li>
	<li>Finally, rinse the slides in formamide solution for 10 min at 4 &#176;C.</li>
</ol>
4. Hybridization
<ol>
	<li>Prepare the antisense and sense probes
	<ol>
		<li>Use hybridization buffer to dilute RNA antisense or sense probes in the 1.5 mL nuclease-free tubes. In this experiment, for all of the antisense and sense probes (LmigOR1, LmigOR2, and LmigOrco), add 1 &#181;L of probe to 99 &#181;L hybridization buffer per slide. Generally, the use of 1:100 dilutions of antisense probes produces significant positive signals and low backgrounds using this protocol.</li>
		<li>For chromogenic detection, dilute DIG-labeled probes individually.</li>
		<li>For TSA detection, dilute DIG-labeled LmigOR1 with biotin-labeled LmigOrco probes or DIG-labeled LmigOR2 with biotin-labeled LmigOR1 probes&#160;together in the hybridization buffer.</li>
		<li>Heat the dilutions for 10 min at 65 &#176;C and put them on ice for at least 5 min.</li>
	</ol>
	</li>
	<li>Hybridization
	<ol>
		<li>Drain the slides and add 100 &#181;L of diluted antisense and sense probes (Step 4.1) to the tissue sections. Then, place coverslips (24 mm x 50 mm; nuclease-free) on the tissue sections.</li>
		<li>Place the covered slides horizontally into a humid box (&#8764;300 &#160;x 180 &#160;x 50 mm3; Figure 2) and incubate at 55 &#176;C for 22 h. Add formamide solution or 1X PBS to the bottom of the box to keep the environment moist, but do not submerge the slides in the liquid.</li>
	</ol>
	</li>
	<li>Washing and blocking.
	<ol>
		<li>After hybridization, remove the coverslips carefully. Wash the slides twice for 30 min in 0.1x Saline Sodium Citrate (SSC) at 60 &#176;C. 
		NOTE: Washing means putting the slides in a slide holder that is then placed into a plastic container and gently agitated on a rocker (&#65374;50 rpm; Figure 2).</li>
		<li>Rinse the slides in 1x Tris-Buffered Saline (TBS) for 30 s.</li>
		<li>Add 1 mL of 1% blocking reagent in TBS supplemented with 0.03% Triton X-100 on each slide, and incubate for 30 min. Then, discard the blocking solution.</li>
	</ol>
	</li>
	<li>Immunohistochemistry.
	<ol>
		<li>For chromogenic detection, use blocking reagent in TBS to dilute 750 units (U)/mL anti-digoxigenin AP-conjugated antibody to 1.5 U/mL AP solution. Add 100 &#181;L of AP solution per covered (24 mm x 50 mm) slide.</li>
		<li>For TSA detection, use blocking reagent in TBS to dilute 750 U/mL of anti-digoxigenin AP- conjugated antibody and anti-biotin streptavidin HRP- conjugated antibody to the AP/HRP solution. Add 100 &#181;L of AP/HRP solution per covered (24 mm x 50 mm) slide.</li>
		<li>Incubate the slides in a humid box (Figure 2) for 60 min at 37 &#176;C. Use the formamide solution or 1X PBS to keep box moist but not soggy.</li>
	</ol>
	</li>
</ol>
5. Staining
<ol>
	<li>Remove the coverslips carefully. Wash the slides three times for 5 min in 1x TBS supplemented with 0.05% Tween-20. Rinse the slides in DAP-buffer (chromogenic detection: pH 9.5; TSA detection: pH 8.0) for 5 min.</li>
	<li>Staining (chromogenic detection)
	<ol>
		<li>Add 100 &#181;L of NBT (375 &#181;g/mL)/BCIP (188 &#181;g/mL) substrate solution (diluted in DAP; pH 9.5) to every slide. Carefully place coverslips (24&#160;x 50 mm) onto the slides.</li>
		<li>Incubate the slides in a humid box with substrate solution for 10 min - O/N at 37 &#176;C. 
		NOTE: Check the development by looking at the slides from time to time under the microscope.</li>
		<li>When the development is ready, stop the reaction by transferring the slides into water.</li>
	</ol>
	</li>
	<li>Staining (TSA detection)
	<ol>
		<li>Use a syringe to move the HNPP (100 &#181;g/mL)/Fast Red (250 &#181;g/mL) substrate from the syringe filter (0.22 &#181;m; Figure 2).</li>
		<li>Add 100 &#181;L of HNPP/Fast Red substrate per slide covered with a coverslip (24&#160;x 50 mm). Incubate the slides for 30 min with HNPP/Fast Red substrate at RT.</li>
		<li>Remove the coverslips carefully, and wash the slides three times for 5 min in 1X TBS supplemented with 0.05% Tween-20.</li>
		<li>Use 100 &#181;L of TSA substrate/covered (24&#160;x 50 mm2) slide. Incubate the slides with TSA substrate for 10 min at RT.</li>
		<li>Remove the coverslips carefully, and wash the slides three times for 5 min in 1x TBS supplemented with 0.05% Tween-20.</li>
	</ol>
	</li>
	<li>Embed the slides in PBS/glycerol (1:3). 
	NOTE: After finishing these procedures, the slides should be observed as soon as possible because the fluorescent signal will quench quickly.</li>
</ol>
6. Observation
<ol>
	<li>Chromogenic detection
	<ol>
		<li>Observe tissue sections using an optical microscope. Choose the 10X, 20X, and 40X objective lenses to observe the results of chromogenic detection.</li>
		<li>Use DP-BSW software to analyze the results and capture the images.</li>
	</ol>
	</li>
	<li>TSA detection
	<ol>
		<li>Observe tissue sections using a confocal microscope. DIG-labeled genes should be observed under 543 nm light, presenting a red color, and biotin-labeled genes should be observed under 488 nm light, presenting a green color. When they emerge, they present yellow color.</li>
		<li>Choose the 20X and 40X objective lenses to observe the results of TSA detection, respectively.</li>
		<li>Use FV1000 software to analyze the results and capture the images.</li>
	</ol>
	</li>
</ol>

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Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.</a> Histochemistry. 98 (4), 217-228 (1992).</li><li>Leary, J. J., Brigati, D. J., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+colorimetric+method+for+visualizing+biotin-labeled+DNA+probes+hybridized+to+DNA+or+RNA+immobilized+on+nitrocellulose:+Bio-blots.">Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.</a> Proc Natl Acad Sci U S A. 80 (13), 4045-4049 (1983).</li><li>Hallem, E. A., Ho, M. G., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+antenna.">The molecular basis of odor coding in the Drosophila antenna.</a> Cell. 117 (7), 965-979 (2004).</li><li>Jones, W. D., Nguyen, T. A., Kloss, B., Lee, K. J., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+conservation+of+an+insect+odorant+receptor+gene+across+250+million+years+of+evolution.">Functional conservation of an insect odorant receptor gene across 250 million years of evolution.</a> Curr Biol. 15 (4), R119-R121 (2005).</li></ol>

The authors have nothing to disclose or any other conflicts of interest.

It is hard to perform RNA ISH to localize OR genes in insect antennae because the expression levels of OR genes, except ORco, are very low and preserving histological slices of insect antennae is very difficult. In addition, TSA detection is also very tricky. To address these problems, the following measures should be taken. The antennae are selected from newly molting adult locusts that have thin and soft antennal cuticles, which maintain their morphology on the slide. The frozen samples are sectioned into 12 &#181;m-thick slices. Highly sensitive and specific biotin- and DIG-labeled probes are used. Detergents, such as Tween-20 and Triton X-100, are used to decrease the background in many steps. In TSA detection, a highly expressed gene, relative to another lowly expressed gene, should be labeled with biotin-16-UTP. The slides should be observed as soon as possible because the fluorescent signals will quench quickly.
DIG- and biotin-labeled probes have the advantages of longer shelf lives, higher signal to noise ratios and better cellular resolutions than radioactive probes18,19. The protocol presented in this paper has some advantages over whole mount in situ hybridization. This protocol easily identifies the localizations of genes at the cellular level but cannot be easily used to investigate gene distribution patterns, which is more readily performed with whole mount in situ hybridization.
To identify an OR gene, two approaches were taken. One was the chromogenic detection in consecutive sections, and the other was fluorescent detection in one section. ORco is co-expressed with a specific OR as an ORN marker10,20,21. Cells expressing LmigOR1 or LmigOR2 were both located to clusters of LmigORco-expressing cells that unambiguously verified them as ORs (Figure 4 and Figure 6). Using the same approaches, we found that LmigOR1 and LmigOR2 are not co-expressed in one sensillum. The results of these two approaches are corroborative.
This protocol was also successfully used to localize LmigORco, SgreORco, SgreIR8a and SgreIR25a in locust antennae10,14. Recently LmigOR3 was localized to trichoid sensilla neurons in L. migratoria using the same protocol15. This protocol was used to localize two sensory neural membrane proteins SgreSNMP1 and SgreSNMP2 in the antennae of S. gregaria16. Thus, this protocol reliably localized chemosensory-related genes in locust antennae, which not only verified these candidate genes, but also localized these genes to specific cells housed in different types of sensilla.
In conclusion, this highly effective protocol of RNA ISH is specifically described to localize OR genes, as well as other genes expressed at low levels, in insect antennae.

Insect antennae, which are the most important chemosensory organs, are covered with many hair-like structures &#8211; called sensilla &#8211; that are innervated by Olfactory Receptor Neurons (ORNs). On the membrane of insect ORNs, Odorant Receptors (ORs), a type of protein containing seven transmembrane domains, are expressed with a coreceptor (ORco) to form a heteromer that functions as an odorant-gated ion channel1,2,3. Different ORs respond to different combinations of chemical compounds4,5,6.
Locusts (Locusta migratoria) mainly rely on olfactory cues to trigger important behaviors7. Locust ORs are key factors for understanding molecular olfactory mechanisms. Localizing a specific locust OR gene to the neuron of a morphologically specific sensillum type by RNA In Situ&#160;Hybridization (RNA ISH) is the first step in exploring the ORs function.
RNA ISH uses a labeled complementary RNA probe to measure and localize a specific RNA sequence in section of tissue, cells or whole mounts in situ, providing insights into physiological processes and disease pathogenesis. Digoxigenin-labeled (DIG-labeled) and biotin-labeled RNA probes have been widely used in RNA hybridization. RNA labeling with digoxigenin-11-UTP or biotin-16-UTP can be prepared by in vitro transcription with SP6 and T7 RNA polymerases. DIG- and biotin-labeled RNA probes have the following advantages: non-radioactive; safe; stable; highly sensitive; highly specific; and easy to produce using PCR and in vitro transcription. DIG- and biotin-labeled RNA probes can be chromogenically and fluorescently detected. DIG-labeled RNA probes can be detected with anti-digoxigenin Alkaline Phosphatase (AP)-conjugated antibodies that can be visualized either with the highly sensitive chemiluminescent substrates nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate toluidine salt (NBT/BCIP) using an optical microscope or with 2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate (HNPP) coupled with 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (Fast Red) using a confocal microscope. Biotin-labeled RNA probes can be detected with anti-biotin streptavidin Horse Radish Peroxidase (HRP)-conjugated antibodies that can be visualized with fluorescein-tyramides using a confocal microscope. Thus, double-color fluorescent in situ hybridization can be performed to detect two target genes in one slice using DIG- and biotin-labeled RNA probes.
RNA ISH with DIG- and/or biotin-labeled probes has been successfully used to localize olfactory-related genes, such as OR, ionotropic receptor, odorant-binding protein and sensory neuron membrane protein, in insect antennae of, but not limited to, Drosophila melanogaster, Anopheles gambiae, L. migratoria and the desert locust Schistocera gregaria8,9,10,11,12,13,14,15,16. However, there are two substantial challenges when performing RNA ISH for insect ORs: (1) OR genes (except ORco) are expressed at low levels and only in a few cells, making signal detection very difficult, and (2) preserving insect tissue for histology, such that the morphology is preserved and the background noise is low, can be challenging. In this paper a detailed and effective protocol describing RNA ISH for localizing OR genes in insect antennae is presented, including both chromogenic and Tyramide Signal Amplification (TSA) detection.

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2&#215;TSINGKETM Master Mix</td>
			<td>TSINGKE, China</td>
			<td>TSE004</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free H2O</td>
			<td>TIANGEN, China</td>
			<td>RT121-02</td>
			<td></td>
		</tr>
		<tr>
			<td>REGULAR AGAROSE G-10</td>
			<td>BIOWEST, SPAIN</td>
			<td>91622</td>
			<td></td>
		</tr>
		<tr>
			<td>Binding buffer</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance buffer</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash solution</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>T Vector</td>
			<td>Promega, USA</td>
			<td>A362A</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega, USA</td>
			<td>M180A</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>TIANGEN, China</td>
			<td>CB101</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma, USA</td>
			<td>A-6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Inalco, USA</td>
			<td>1758-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside</td>
			<td>SBS Genetech, China</td>
			<td>GX1-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nco I</td>
			<td>BioLabs, New England</td>
			<td>R0193S</td>
			<td></td>
		</tr>
		<tr>
			<td>Spe I</td>
			<td>BioLabs, New England</td>
			<td>R0133M</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG RNA Labeling Kit</td>
			<td>Roche, Switzerland</td>
			<td>11175025910</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin RNA Labeling Kit</td>
			<td>Roche, Switzerland</td>
			<td>11685597910</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>DIG RNA Labeling Kit, Roche, Switzerland</td>
			<td>11175025910</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sinopharm, China</td>
			<td>10012718</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm, China</td>
			<td>10009257</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>BEIJING CHEMICAL REGENTS COMPANY, China</td>
			<td>10000292</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura Finetek Europe, Zoeterwoude, Netherlands</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>TINA JIN HAO YANG BIOLOGCAL MANUFACTURE CO., LTE, China</td>
			<td>FISH0010</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sinopharm, China</td>
			<td>80070591</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex</td>
			<td>Millipore, USA</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>AMRESCO, USA</td>
			<td>0777-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitroblue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine salt</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm, China</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>0.04mol/L Tris-Base</td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>BEIJING CHEMICAL REGENTS COMPANY, China</td>
			<td>10000292</td>
			<td>0.12%acetic acid</td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>Sigma, USA</td>
			<td>03677</td>
			<td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>10g/L NaCl</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>MERCK, Germany</td>
			<td>VM335231</td>
			<td>10g/L Peptone from casein (Tryptone)</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>MERCK, Germany</td>
			<td>VM361526</td>
			<td>5g/L Yeast extract</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>10g/L NaCl</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>MERCK, Germany</td>
			<td>VM335231</td>
			<td>10g/L Peptone from casein (Tryptone)</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>MERCK, Germany</td>
			<td>VM361526</td>
			<td>5g/L Yeast extract</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>WISENT ING, Canada</td>
			<td>800-010-CG</td>
			<td>15g/L Agar Bacteriological Grade</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>8.5%NaCl</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sigma, USA</td>
			<td>V900041-500G</td>
			<td>14mM KH2PO4</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sigma, USA</td>
			<td>V900268-500G</td>
			<td>80mM Na2HPO4</td>
		</tr>
		<tr>
			<td>10&#215;Tris buffered saline (pH7.5)</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>1M Tris-Base</td>
		</tr>
		<tr>
			<td>10&#215;Tris buffered saline (pH7.5)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>1.5M NaCl</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>100mM Tris-Base</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>100mM NaCl</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sigma, USA</td>
			<td>V900020-500G</td>
			<td>50mM MgCl2&#183;6H2O</td>
		</tr>
		<tr>
			<td>20&#215;saline-sodium citrate (pH7.0)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>3M NaCl</td>
		</tr>
		<tr>
			<td>20&#215;saline-sodium citrate (pH7.0)</td>
			<td>Sigma, USA</td>
			<td>V900095-500G</td>
			<td>0.3M Na-Citrate</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution (pH9.5)</td>
			<td>Sigma, USA</td>
			<td>V900894-100G</td>
			<td>4% paraformaldehyde</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution (pH9.5)</td>
			<td>Sigma, USA</td>
			<td>V900182-500G</td>
			<td>0.1M NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Buffer (pH10.2)</td>
			<td>Sigma, USA</td>
			<td>V900182-500G</td>
			<td>80mM NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Buffer (pH10.2)</td>
			<td>Sigma, USA</td>
			<td>S7795-500G</td>
			<td>120mM Na2CO3</td>
		</tr>
		<tr>
			<td>Formamide Solution (pH10.2)</td>
			<td>MPBIO, USA</td>
			<td>FORMD002</td>
			<td>50% Deionized Formamide</td>
		</tr>
		<tr>
			<td>Formamide Solution (pH10.2)</td>
			<td></td>
			<td></td>
			<td>5&#215;saline-sodium citrate</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1% Blot</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td>AMRESCO, USA</td>
			<td>0694-500ML</td>
			<td>0.03% Triton X-100</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td></td>
			<td></td>
			<td>1&#215;Tris buffered saline</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase solution</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase solution</td>
			<td></td>
			<td></td>
			<td>Blocking Buffer in Tris buffered saline</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>1% anti-biotin streptavidin horse radish peroxidase- conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td></td>
			<td></td>
			<td>Blocking Buffer in Tris buffered saline</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>MPBIO, USA</td>
			<td>FORMD002</td>
			<td>50% Deionized Formamide</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td></td>
			<td></td>
			<td>2&#215;saline-sodium citrate</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>Sigma, USA</td>
			<td>D8906-50G</td>
			<td>10% dextran sulphate</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>invitrogen, USA</td>
			<td>AM7119</td>
			<td>20 &#181;g/ml yeast t-RNA</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>Sigma, USA</td>
			<td>D3159-10G</td>
			<td>0.2 mg/ml herring sperm DNA</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td>Roche, Switzerland</td>
			<td>11758888001</td>
			<td>1% 2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate (10mg/ml)</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td>Roche, Switzerland</td>
			<td>11758888001</td>
			<td>1% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (25mg/ml)</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td></td>
			<td></td>
			<td>Detection Buffer</td>
		</tr>
		<tr>
			<td>Tyramide signal amplification substrate</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>2% fluorescein-tyramides</td>
		</tr>
		<tr>
			<td>Tyramide signal amplification substrate</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>Amplification Diluent</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Instrument</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Leica, Nussloch, Germany</td>
			<td>Jung CM300 cryostat</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo SCIENTIFIC, USA</td>
			<td>NANODROP 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>Olympus IX71microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>Olympus BX45 confocal microscope</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

With chromogenic detection, a small subset of the antennal cells in every adult antennal section was denoted by the DIG-labeled LmigOR1 and LmigOR2 antisense probes (Figure 3). RNA ISH on consecutive sections to localize LmigOR1 and LmigOR2 showed that antennal cells expressing the two genes were located in ORN clusters expressing LmigORco, indicating that the putative LmigOR1 and LmigOR2 were actually expressed in ORNs (Figure 4a-4d). Occasionally, labeled dendritic-like structures were visualized (Figure 4e-4f). LmigOR1 and LmigOR2 were both localized to neurons in the basiconic sensilla (Figure 5a-5b), but they were not co-expressed in individual sensilla, indicating that they were present in different basiconic sensillium subtypes (Figure 5c-5d).
In TSA detection, double-color fluorescent in situ hybridization showed that LmigOR1-expressing cells (red color) were located to ORN clusters expressing LmigORco (green color), indicating that the putative LmigOR1 was expressed in ORNs (Figure 6a). A close view of the boxed areas in Figure 6a are shown in Figure 6b. LmigOR1- expressing neurons (green color, Figure 7a) and LmigOR2-expressing neuron (red color, Figure 7b) were located to different basiconic sensillium subtypes (Figure 7c).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55924/55924fig1.jpg" /> 
Figure 1: Preparation of Insect Antennal Sections for RNA In Situ Hybridization. (a) The freezing microtome; (b-c) The samples are embedded in freezing O.C.T. Compound. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55924/55924fig2.jpg" /> 
Figure 2: Experimental Items for RNA In Situ&#160;Hybridization. (a) The slide holder and the plastic container. (b) The humid box. (c) The rocker. (d) The membrane filter. (e) Confocal microscope. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55924/55924fig3.jpg" /> 
Figure 3: Cellular Localization of LmigOR1 &#38; LmigOR2 in Olfactory Organs. (a) Overview of LmigOR1-expressing cells in a locust antennal segment. (b) Overview of LmigOR2-expressing cells in a locust antennal segment. Arrowheads indicate cells expressing LmigOR1 (a) and LmigOR2 (b). Scale bars = 100 &#181;m (a and b). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55924/55924fig4.jpg" /> 
Figure 4: Neuronal Identity of Antennal Cells Expressing LmigORs by Chromogenic Detections. (a-b) The labeling pattern of LmigOR1 (a) and LmigORco (b) antisense probe on consecutive sections of locust antenna. (c-d) The labeling pattern of LmigORco (c) and LmigOR2 (d) antisense probe on consecutive sections of locust antenna. (e-f) Illustration of occasionally labeled dendritic-like structures (indicated by red arrows). Scale bars = 50 &#181;m (a-d); 20 &#181;m (e and f). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55924/55924fig5.jpg" /> 
Figure 5: LmigOR1 &#38; LmigOR2&#160;are Expressed in ORNs Housed in Basiconic Sensilla. (a-b) Basiconic sensillum housed ORNs expressing LmigOR1 (a) and LmigOR2 (b). (c-d) The expression of LmigOR1 and LmigOR2 in distinct subset of antennal ORNs was verified on consecutive sections (c-d). Arrowheads denote antennal cells expressing LmigOR1 (a, c) and LmigOR2 (b, d). Ba: basiconic sensillum. Scale bars = 20 &#181;m (a and b); 50 &#181;m (c and d). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55924/55924fig6.jpg" /> 
Figure 6: Neuronal Identity of Antennal Cell Expressing LmigOR1 by TSA Detections. (a) Two-color in situ hybridization was performed on longitudinal antennal section to illustrate the expression of LmigOR1 (Red) and LmigORco (Green). Localization of LmigOR1-expressing cells in cell clusters expressing LmigORco confirmed its neural identity. (b) Close view of boxed areas in a. Occasionally labeled dendritic like structures were indicated by arrow. Circled areas indicate ORNs cluster expressing LmigORco and sharing the same sensillum. Scale bars = 50 &#181;m (a); 20 &#181;m (b). Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55924/55924fig7.jpg" /> 
Figure 7: LmigOR1 &#38; LmigOR2 were Located to Different Basiconic Sensilla ORNs by TSA Detection. Fluorescent signals were visualized using detection systems, indicating LmigOR1-labeled neurons by green fluorescence (a) and LmigOR2 positive cells by red fluorescence (b) were expressed in different basiconic sensilla subtypes (c). Arrowheads denote antennal cells expressing LmigOR1 and LmigOR2. Scale bars = 20 &#181;m. Figures were adapted from Xu et al.12 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Sequences</td>
		</tr>
		<tr>
			<td>Lmig OR1-probe-s</td>
			<td>5&#8217;-AAGGGGTGGGAGACGGCCTG-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig OR1-probe-as</td>
			<td>5&#8217;-CAGCTCCTCCCCAACGACAGC-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig OR2-probe-s</td>
			<td>5&#8217;-ATGGGTGAGCGTGGAGAGGC-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig OR2-probe-as</td>
			<td>5&#8217;-GGTCATCGCTGTGGACGTGG-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig Orco-probe-s</td>
			<td>5&#8217;-CTCGTCTGACAGCGTAACTCAC-3&#8217;</td>
		</tr>
		<tr>
			<td>Lmig Orco-probe-as</td>
			<td>5&#8217;-AAGACGCAGAAGAGGAAGACCT-3&#8217;</td>
		</tr>
	</tbody>
</table>
Table 1: The Sequences of the PCR Primers.

Watch this Scientific Journal Video about Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization at JoVE.com

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.

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 ...

localization-odorant-receptor-genes-locust-antennae-rna-situ

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.0K Views.  China Agricultural University. 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.

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

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 ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

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 ...

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 ...

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 ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.