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Method Article

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

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DOI:

10.3791/55924

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July 13th, 2017

 ,  , 
1Department of Entomology, China Agricultural University, 2Bio-tech Research Center, Shandong Academy of Agricultural Sciences

In This Article

Summary

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.

Abstract

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' 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.

Introduction

Insect antennae, which are the most important chemosensory organs, are covered with many hair-like structures – called sensilla – 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 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'-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.

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Protocol

NOTE: To limit RNA degradation, prepare solutions using wet-autoclaved distilled water (at 121 °C for 60 min) and also wet-autoclave materials.

1. Preparation of RNA ISH Antisense and Sense Probes

  1. Target gene amplification and purification
    1. 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.
      1. Use a 100 µL final reaction volume, containing 50 µL of 2x reaction mix, 4 µL each of sense and antisense primers (Table 1), 1 µL of plasmid template, and 41 µL of RNase-free H2O. Use the following PCR protocol: 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, followed by 72 °C for 10 min.
      2. 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.
      3. Run PCR products on 1.2% agarose gels in 1x Tris-acetate-EDTA buffer and visualize using ethidium bromide staining.
    2. Extract these PCR products using a gel extraction kit.
      1. Following electrophoresis, excise DNA bands from the gel and place the gel slices in a 1.5 mL tube. Then, add 100 µL of binding buffer (Buffer PN) per 0.1 g of gel slice. Vortex and incubate at 50 °C until the gel slice is completely dissolved.
      2. Insert a CA2 adsorption column into the collection tube and add 500 µL 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.
      3. Transfer the dissolved gel mixture to the adsorption column assembly and incubate 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.
      4. Add 600 µL 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.
      5. Empty the collection tube and recentrifuge the column assembly at 12,400 x g for 2 min to allow evaporation of any residual ethanol.
      6. 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 µL) of nuclease-free water.
      7. Incubate at RT for 2 min. Centrifuge at 12,400 x g for 2 min. Discard adsorption column and store the DNA at 4 °C or -20 °C.
  2. Construct the recombinant plasmids
    1. Individually ligate the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2 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 µL reaction: 5 µL of 2x ligation buffer, 1 µL of T vector, 3 µL (∼100 ng) of the inserted gene's DNA and 1 µL of T4 DNA ligase, and incubate O/N at 4 °C.
    2. Add 5 µL of each recombinant plasmid containing the 387 bp fragment of LmigOR1, 1,303 bp fragment of LmigOR2 and 1,251 bp fragment of LmigORco separately into 50 µL of competent Escherichia coli DH5α cells in sterile 1.5 mL tubes.
      1. Mix gently and put the tubes into ice for 30 min and then incubate them for 90 s at 42 °C. Transfer them back into ice for 3 min.
      2. Add 450 µL 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 °C to restore the E. coli DH5α.
      3. Take a 100 µL aliquot of each transformant and use them to inoculate LB solid substrate plates containing 50 µg/mL ampicillin, 24 µg/mL isopropyl β-D-1-thiogalactopyranoside (IPTG), and 40 µg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal).
      4. Incubate these plates in a constant incubator at 37 °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' insert directions.
  3. Select restriction endonucleases to linearize recombinant plasmids
    1. 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.
    2. 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'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.
  4. Purifying the linearized recombinant plasmids
    1. Digest 20 µg 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 µL reaction that contains 10 µL of 10x buffer, 40 µL of 0.5 µg/µL plasmid, 3 µL of Nco I and 47 µL of RNase-free H2O, followed by a 3 h incubation at 37 °C.
    2. Similarly, digest 20 µg of each of these three recombinant plasmids using the Spe I restriction endonuclease.
    3. 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 µg/µL.
  5. Prepare antisense and sense probes
    1. 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 µL final volume, containing 2 µL of 10x NTP mix, 2 µL of 10X transcription buffer, 1 µL of RNase inhibitor, 2 µL of T7 or SP6 RNA polymerase, 4 µL of 0.5 µg/µL linearized plasmid and 9 µL RNase-free H2O, followed by a 3 h incubation at 37 °C.
    2. Incubate the DIG- and biotin-labeled antisense and sense probes for 30 min with 1 µL DNase at 37 °C. Add 2.5 µL of 5 M LiCl and 75 µL of absolute ethanol, then incubate the reactions for 30 min at -70 °C.
    3. Centrifuge at 15,000 x g for 30 min at 4 °C. Carefully decant the supernatant. Add 150 µL 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).
    4. Centrifuge at 15,000 x g for 30 min at 4 °C again and air-dry the pellet for 5-10 min at RT. Add 25 µL of RNase-free H2O to dissolve the RNA antisense and sense probes.
    5. If the length of the inserted gene is longer than 1 kb, subsequently fragment RNA antisense and sense probes to an average length of ～300 bp by incubation in a bicarbonate-carbonate buffer solutions. Perform the reaction in a 50 µL final volume, containing 25 µL of the RNA probe and 25 µL of the bicarbonate-carbonate buffer solution (80 mM NaHCO3, 120 mM Na2CO3, pH 10.2) at 60 °C. The hydrolysis time required is given by a previously published formula17.
    6. Add 5 µL 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
    7. Finally, add 250 µL of hybridization buffer and store at -80 °C.

2. Preparation of Cryostat Sections

  1. Precool the freezing microtome to -24 °C.
  2. Select new molting adult locusts that are active and have intact antennae. Cut the antennae into 2-3 mm pieces using sterile razors.
  3. 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 °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 °C again for at least 10 min (Figure 1).
    NOTE: Avoid getting bubbles in the compound.
  4. Fix the holder with the samples into the freezing microtome, then section the frozen samples into 12 µm-thick slices at -24 °C.
  5. Thaw mount the slices one by one on slides (25  x 75 mm; nuclease-free) and air dry for 10 min.

3. Fixing Sections

  1. After preparing cryostat sections, put the slides in a plastic container (～100  x 40  x 80 mm), and fix the tissues by incubating the slides in 4% paraformaldehyde solution (PFA) for 30 min at 4 °C.
  2. Wash the slides in 1X Phosphate-Buffered Saline (PBS) for 1 min.
  3. To eliminate the alkaline proteins, transfer the slides to 0.2 M HCl for 10 min.
  4. To eliminate the surface protein of nucleic acid, transfer the slides to 1XPBS with 1% Triton X-100 for 2 min.
  5. Wash the slides twice for 30 s in 1x PBS.
  6. Finally, rinse the slides in formamide solution for 10 min at 4 °C.

4. Hybridization

  1. Prepare the antisense and sense probes
    1. 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 µL of probe to 99 µL hybridization buffer per slide. Generally, the use of 1:100 dilutions of antisense probes produces significant positive signals and low backgrounds using this protocol.
    2. For chromogenic detection, dilute DIG-labeled probes individually.
    3. For TSA detection, dilute DIG-labeled LmigOR1 with biotin-labeled LmigOrco probes or DIG-labeled LmigOR2 with biotin-labeled LmigOR1 probes together in the hybridization buffer.
    4. Heat the dilutions for 10 min at 65 °C and put them on ice for at least 5 min.
  2. Hybridization
    1. Drain the slides and add 100 µL 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.
    2. Place the covered slides horizontally into a humid box (∼300  x 180  x 50 mm3; Figure 2) and incubate at 55 °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.
  3. Washing and blocking.
    1. After hybridization, remove the coverslips carefully. Wash the slides twice for 30 min in 0.1x Saline Sodium Citrate (SSC) at 60 °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 (～50 rpm; Figure 2).
    2. Rinse the slides in 1x Tris-Buffered Saline (TBS) for 30 s.
    3. 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.
  4. Immunohistochemistry.
    1. 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 µL of AP solution per covered (24 mm x 50 mm) slide.
    2. 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 µL of AP/HRP solution per covered (24 mm x 50 mm) slide.
    3. Incubate the slides in a humid box (Figure 2) for 60 min at 37 °C. Use the formamide solution or 1X PBS to keep box moist but not soggy.

5. Staining

  1. 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.
  2. Staining (chromogenic detection)
    1. Add 100 µL of NBT (375 µg/mL)/BCIP (188 µg/mL) substrate solution (diluted in DAP; pH 9.5) to every slide. Carefully place coverslips (24 x 50 mm) onto the slides.
    2. Incubate the slides in a humid box with substrate solution for 10 min - O/N at 37 °C.
      NOTE: Check the development by looking at the slides from time to time under the microscope.
    3. When the development is ready, stop the reaction by transferring the slides into water.
  3. Staining (TSA detection)
    1. Use a syringe to move the HNPP (100 µg/mL)/Fast Red (250 µg/mL) substrate from the syringe filter (0.22 µm; Figure 2).
    2. Add 100 µL of HNPP/Fast Red substrate per slide covered with a coverslip (24 x 50 mm). Incubate the slides for 30 min with HNPP/Fast Red substrate at RT.
    3. Remove the coverslips carefully, and wash the slides three times for 5 min in 1X TBS supplemented with 0.05% Tween-20.
    4. Use 100 µL of TSA substrate/covered (24 x 50 mm2) slide. Incubate the slides with TSA substrate for 10 min at RT.
    5. Remove the coverslips carefully, and wash the slides three times for 5 min in 1x TBS supplemented with 0.05% Tween-20.
  4. 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.

6. Observation

  1. Chromogenic detection
    1. Observe tissue sections using an optical microscope. Choose the 10X, 20X, and 40X objective lenses to observe the results of chromogenic detection.
    2. Use DP-BSW software to analyze the results and capture the images.
  2. TSA detection
    1. 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.
    2. Choose the 20X and 40X objective lenses to observe the results of TSA detection, respectively.
    3. Use FV1000 software to analyze the results and capture the images.

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Results

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 act...

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Discussion

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 &...

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Disclosures

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

Acknowledgements

This work is supported by a grant 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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Materials
2×TSINGKETM Master MixTSINGKE, ChinaTSE004
RNase-free H2OTIANGEN, ChinaRT121-02
REGULAR AGAROSE G-10BIOWEST, SPAIN91622
Binding bufferTIANgel Midi Purification Kit, TIANGEN, ChinaDP209-02
Balance bufferTIANgel Midi Purification Kit, TIANGEN, ChinaDP209-02
Wash solutionTIANgel Midi Purification Kit, TIANGEN, ChinaDP209-02
T VectorPromega, USAA362A
T4 DNA LigasePromega, USAM180A
Escherichia coli DH5αTIANGEN, ChinaCB101
AmpicillinSigma, USAA-6140
Isopropyl β-D-1-thiogalactopyranosideInalco, USA1758-1400
5-bromo-4-chloro-3-indolyl-β-D-galactopyranosideSBS Genetech, ChinaGX1-500
Nco IBioLabs, New EnglandR0193S
Spe IBioLabs, New EnglandR0133M
DIG RNA Labeling KitRoche, Switzerland11175025910
Biotin RNA Labeling KitRoche, Switzerland11685597910
DNaseDIG RNA Labeling Kit, Roche, Switzerland11175025910
LiClSinopharm, China10012718
EthanolSinopharm, China10009257
Acetic acidBEIJING CHEMICAL REGENTS COMPANY, China10000292
Tissue-Tek O.C.T. CompoundSakura Finetek Europe, Zoeterwoude, Netherlands4583
SlidesTINA JIN HAO YANG BIOLOGCAL MANUFACTURE CO., LTE, ChinaFISH0010
HClSinopharm, China80070591
MillexMillipore, USASLGP033RS
Tween 20AMRESCO, USA0777-500ML
Nitroblue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine saltRoche, Switzerland11175041910
GlycerolSinopharm, China10010618
NameCompanyCatalog NumberComments
Solutions
1×Tris-acetate-EDTASigma, USAV900483-1KG0.04 mol/L Tris-Base
1×Tris-acetate-EDTABEIJING CHEMICAL REGENTS COMPANY, China100002920.12% acetic acid
1×Tris-acetate-EDTASigma, USA03677Ethylenediaminetetraacetic acid disodium salt (EDTA)
Luria-Bertani (LB) liquid mediumSinopharm, China1001939210 g/L NaCl
Luria-Bertani (LB) liquid mediumMERCK, GermanyVM33523110 g/L Peptone from casein (Tryptone)
Luria-Bertani (LB) liquid mediumMERCK, GermanyVM3615265 g/L Yeast extract
LB solid substrate plateSinopharm, China1001939210 g/L NaCl
LB solid substrate plateMERCK, GermanyVM33523110 g/L Peptone from casein (Tryptone)
LB solid substrate plateMERCK, GermanyVM3615265 g/L Yeast extract
LB solid substrate plateWISENT ING, Canada800-010-CG15 g/L Agar Bacteriological Grade
10×Phosphate-Buffered Saline (PBS, pH 7.1)Sinopharm, China100193928.5% NaCl
10×Phosphate-Buffered Saline (PBS, pH 7.1)Sigma, USAV900041-500G14 mM KH2PO4
10×Phosphate-Buffered Saline (PBS, pH 7.1)Sigma, USAV900268-500G80 mM Na2HPO4
10×Tris-Buffered Saline (TBS, pH 7.5)Sigma, USAV900483-1KG1 M Tris-Base
10×Tris-Buffered Saline (TBS, pH 7.5)Sinopharm, China100193921.5 M NaCl
Detection Buffer (DAP)       chromogenic detection pH 9.5       TSA detection pH 8.0Sigma, USAV900483-1KG100 mM Tris-Base
Detection Buffer (DAP)       chromogenic detection pH 9.5       TSA detection pH 8.0Sinopharm, China10019392100 mM NaCl
Detection Buffer (DAP)       chromogenic detection pH 9.5       TSA detection pH 8.0Sigma, USAV900020-500G50 mM MgCl2·6H2O
20×saline-sodium citrate (pH 7.0)Sinopharm, China100193923 M NaCl
20×saline-sodium citrate (pH 7.0)Sigma, USAV900095-500G0.3 M Na-Citrate
4% paraformaldehyde solution (PFA, pH 9.5)Sigma, USAV900894-100G4% paraformaldehyde
Sodium Carbonate BufferSigma, USAV900182-500G0.1 M NaHCO3
Sodium Carbonate Buffer (pH 10.2)Sigma, USAV900182-500G80 mM NaHCO3
Sodium Carbonate Buffer (pH 10.2)Sigma, USAS7795-500G120 mM Na2CO3
Formamide Solution (pH 10.2)MPBIO, USAFORMD00250% Deionized Formamide
Formamide Solution (pH 10.2)5×saline-sodium citrate
Blocking Buffer in TBSRoche, Switzerland111750419101% Blot
Blocking Buffer in TBSAMRESCO, USA0694-500ML0.03% Triton X-100
Blocking Buffer in TBS1×Tris buffered saline
Alkaline phosphatase solutionRoche, Switzerland111750419101.5 U/mL anti-digoxigenin alkaline phosphatase conjugated antibody
Alkaline phosphatase solutionBlocking Buffer in TBS
Alkaline phosphatase/ horse radish peroxidase solutionRoche, Switzerland111750419101.5 U/mL anti-digoxigenin alkaline phosphatase conjugated antibody
Alkaline phosphatase/ horse radish peroxidase solutionTSA kit, Perkin Elmer, USANEL701A001KT1% anti-biotin streptavidin horse radish peroxidase- conjugated antibody
Alkaline phosphatase/ horse radish peroxidase solutionBlocking Buffer in TBS
Hybridization BufferMPBIO, USAFORMD00250% Deionized Formamide
Hybridization Buffer2×saline-sodium citrate
Hybridization BufferSigma, USAD8906-50G10% dextran sulphate
Hybridization Bufferinvitrogen, USAAM711920 µg/mL yeast t-RNA
Hybridization BufferSigma, USAD3159-10G0.2 mg/mL herring sperm DNA
2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrateRoche, Switzerland117588880011% 2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate (10 mg/mL)
2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrateRoche, Switzerland117588880011% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (25 mg/mL)
2-hydroxy-3-naphtoic acid-2'-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrateDetection Buffer
Tyramide signal amplification substrateTSA kit, Perkin Elmer, USANEL701A001KT2% fluorescein-tyramides
Tyramide signal amplification substrateTSA kit, Perkin Elmer, USANEL701A001KTAmplification Diluent
NameCompanyCatalog NumberComments
Instrument
Freezing microtomeLeica, Nussloch, GermanyJung CM300 cryostat
SpectrophotometerThermo SCIENTIFIC, USANANODROP 2000
Optical microscopeOlympus, Tokyo, JapanOlympus IX71microscope
Confocal microscopeOlympus, Tokyo, JapanOlympus BX45 confocal microscope

References

  1. Sato, K., et al. Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature. 452 (7190), 1002-1006 (2008).
  2. Smart, R., et al. Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins. Insect Biochem Mol Biol. 38 (8), 770-780 (2008).
  3. Wicher, D., et al. Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature. 452 (7190), 1007-1011 (2008).
  4. Carey, A. F., Wang, G., Su, C. Y., Zwiebel, L. J., Carlson, J. R. Odorant reception in the malaria mosquito Anopheles gambiae. Nature. 464 (7258), 66-71 (2010).
  5. Hallem, E. A., Carlson, J. R. Coding of odors by a receptor repertoire. Cell. 125 (1), 143-160 (2006).
  6. Wang, G., Carey, A. F., Carlson, J. R., Zwiebel, L. J. Molecular basis of odor coding in the malaria vector mosquito Anopheles gambiae. Proc Natl Acad Sci U S A. 107 (9), 4418-4423 (2010).
  7. Hassanali, A., Njagi, P. G., Bashir, M. O. Chemical ecology of locusts and related acridids. Annu Rev Entomol. 50, 223-245 (2005).
  8. Schaeren-Wiemers, N., Gerfin-Moser, A. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labeled cRNA probes. Histochemistry. 100 (6), 431-440 (1993).
  9. Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A., Axel, R. A spatial map of olfactory receptor expression in the Drosophila antenna. Cell. 96 (5), 725-736 (1999).
  10. Yang, Y., Krieger, J., Zhang, L., Breer, H. The olfactory co-receptor Orco from the migratory locust (Locusta migratoria) and the desert locust (Schistocerca gregaria): identification and expression pattern. Int J Biol Sci. 8 (2), 159-170 (2012).
  11. Schultze, A., et al. The co-expression pattern of odorant binding proteins and olfactory receptors identify distinct trichoid sensilla on the antenna of the malaria mosquito Anopheles gambiae. PLoS One. 8 (7), e69412(2013).
  12. Xu, H., Guo, M., Yang, Y., You, Y., Zhang, L. Differential expression of two novel odorant receptors in the locust (Locusta migratoria). BMC Neurosci. 14, 50(2013).
  13. Saina, M., Benton, R. Visualizing olfactory receptor expression and localization in Drosophila. Methods Mol Biol. 1003, 211-228 (2013).
  14. Guo, M., Krieger, J., Große-Wilde, E., Mißbach, C., Zhang, L., Breer, H. Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria). Int J Biol Sci. 10 (1), 1-14 (2013).
  15. You, Y., Smith, D. P., Lv, M., Zhang, L. A broadly tuned odorant receptor in neurons of trichoid sensilla in locust, Locusta migratoria. Insect Biochem Mol Biol. 79, 66-72 (2016).
  16. Jiang, X., Pregitzer, P., Grosse-Wilde, E., Breer, H., Krieger, J. Identification and characterization of two "Sensory Neuron Membrane Proteins" (SNMPs) of the desert locust, Schistocerca gregaria (Orthoptera: Acrididae). J Insect Sci. 16 (1), 33(2016).
  17. Angerer, L. M., Angerer, R. C. In situ. hybridization to cellular RNA with radiolabelled RNA probes. In situ hybridization. Wilkinson, D. G. , IRL Press. Oxford. 2(1992).
  18. Komminoth, P., Merk, F. B., Leav, I., Wolfe, H. J., Roth, J. Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate. Histochemistry. 98 (4), 217-228 (1992).
  19. Leary, J. J., Brigati, D. J., Ward, D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 80 (13), 4045-4049 (1983).
  20. Hallem, E. A., Ho, M. G., Carlson, J. R. The molecular basis of odor coding in the Drosophila antenna. Cell. 117 (7), 965-979 (2004).
  21. Jones, W. D., Nguyen, T. A., Kloss, B., Lee, K. J., Vosshall, L. B. Functional conservation of an insect odorant receptor gene across 250 million years of evolution. Curr Biol. 15 (4), R119-R121 (2005).

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Olfactory Receptor NeuronsChemosensillaDouble-Color Fluorescent HybridizationOrco Marker GeneChromogenic DetectionTSA DetectionConfocal Microscopy
Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization
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