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

1. Considerations and preparation of materials

1. Decide if whole-mount or cryo-section of tissue will be appropriate. This protocol is optimized for whole-mount in situ imaging of RNA in the Anopheles mosquito antenna and maxillary palp without cryo-sectioning. If samples are thicker than 5 mm, cryo-sectioning is recommended to enable probe penetration.
2. Identify the genes of interest and copy the sequences including introns and exons from a suitable database. Transcribe the gene sequence into RNA for synthesis.
3. Determine whether to purchase probes from commercial vendors or if probes will be synthesized in the laboratory.
   NOTE: In this study, the in situ probes were purchased from a commercial vendor (see Table of Materials). Alternatively, it can be synthesized as previously reported¹⁰. Reagents used in the study are described in Table 1. Materials required to prepare the reagents are listed in the Table of Materials.

2. Tissue pre-fixation

1. Anesthetize 10-15 adult female or male Anopheles coluzzii mosquitoes (strain N'Gousso), aged 5-10 days post emergence, by collecting them with a mouth aspirator into a paper cup or plastic tube and placing them in a bucket containing ice. A successful cold-induced anesthesia can be confirmed when the mosquitoes are immobile.
2. Decapitate them by removing the heads from the neck region with a pair of forceps. Using the non-dominant hand grab the thorax with forceps and separate the neck from the body with forceps held by the dominant hand. Place heads in a 1.5 mL tube containing 500 µL of chitinase-chymotrypsin dimethyl sulfoxide buffer (CCD buffer) on ice.
   NOTE: Freezing animals can deform the antennae; a quick knockdown on ice is recommended. The mosquito's age during testing may vary depending on the project. It is important to confirm and coordinate their physiological status, such as whether they are blood-fed, starved, or mated. In this study, olfactory appendages were sampled from mosquitoes that had been blood-fed and mated.
3. Pre-heat mosquito heads in CCD buffer at 37 ˚ C on a heat block for 5 min and then transfer the tube containing the mosquito heads to a hybridization oven and rotate at 37 ˚ C. Incubation time in CCD buffer depends on tissue type. For female Anopheles antennae, use 20 min, male antennae require 15 min, while longer incubation time (1 h) is needed for the maxillary palps.
4. Transfer the entire content of the tube into a dissecting watch glass. Gently pour sample into the depression of a watch (dissection) glass. If mosquito heads are stuck inside the tube, use a pipette to add CCD buffer into the tube and rinse out the head.
5. Use forceps to carefully transfer the heads or any antennae/palps detached during incubation and fix in 1 mL of the pre-fixative.
   NOTE: Leftover CCD buffer can be preserved at -20 ˚ C. Buffer may be reused up to 3x. If the pre-fixative turns slightly brownish because of the CCD buffer carryover by the tissues, replace it with another 1 mL of fixative.
6. Rotate heads in pre-fixative for 24 h at 4˚ C using a nutator.
   ​NOTE: Rocking speed for the nutator used in this study was non-adjustable; the default speed set (12 rpm) by the manufacturer was used.

3. Tissue dissection

1. Rinse heads 4x (5 min per wash) with 1 mL of 0.1% PBS-Tween on ice. To avoid sample loss, use a pipette attached to gel loading tip to remove the liquid.
   NOTE: Two quick washes followed by a 10 min long wash, and a final quick wash could be performed instead of step 3.1.
2. Transfer the heads in the tube into a dissecting watch glass. Gently pour sample into the depression of a watch glass. Rinse out mosquito heads that are stuck inside the tube with 0.1% PBS-Tween.
3. Under a dissecting microscope, remove tissues of interest (antennae/palps) from the head with sharp forceps. Hold the posterior part of the head with forceps and grab an antenna with another forceps from the base. Clean forceps with paper moistened with RNAse-free solvent. Remove the palps using the same process.
4. Transfer the antennae and palps with forceps into empty DNA/RNase-free tubes placed on ice. Separate the different tissue parts into pre-labeled different tubes.
5. Dehydrate tissue in 400 µL of solvent containing a mixture of methanol (MeOH 80%) and Dimethyl sulfoxide (DMSO 20%) for 1 h at room temperature.
   NOTE: There is no need to place tissue on a nutator; let it sit on a tube rack at room temperature on the lab bench. It is recommended to use fresh solvent mixture. For a 500 µL stock solution, mix 400 µL of MeOH with 100 µL of DMSO.
6. Replace the dehydrating reagent with 400 µL of absolute (100%) methanol and dehydrate tissues overnight at -20 ˚C. Allow tissues to settle by gravity and use a pipette to remove the liquid.
   ​NOTE: Samples are viable for extended period of dehydration, up to 4 nights have been tested without loss of signal.

4. Tissue post-fixation

1. Rehydrate tissues in a four-step series of graded 400 µL MeOH/PBS-Tween for 10 min on ice. Start with 75% MeOH/25% PBS-Tween followed by 50% MeOH/ 50% PBS-Tween, then 25% MeOH/ 75% PBS-Tween and finally 100% PBS-Tween.
   NOTE: A gel loading tip with a tiny opening can be used to remove the buffer from the tube to avoid losing the tissues. Buffer removal can be done under a dissecting microscope.
2. Wash with 400 µL of phosphate-buffered saline containing 0.1% Tween-20 (PBS-T) for 10 min at room temperature. Wash by placing the sample on a nutator.
3. Make a 20 µg/mL Proteinase-K solution and incubate in 400 µL of Proteinase-K solution for 30 min at room temperature.
   NOTE: Dilute 20 mg/mL Proteinase-K stock (1000x stock solution) i.e., 2 µL in 2 mL of 0.1% PBS-Tween. The left over can be stored in a -20 ˚ C freezer.
4. Stop enzymatic digestion of tissue by washing 2x for 10 min with 400 µL of 0.1% PBS-tween.
5. Add 400 µL of the post-fixative and incubate for 20 min at room temperature. Wash 3x, 15 min per wash, with 400 µL of 0.1% PBS-Tween.

5. Probe hybridization

1. Incubate tissue in 400 µL of probe hybridization buffer for 5 min. Ensure tissue is completely submerged in the buffer by gently pipetting.
2. Prepare for the next step by heating an aliquot of probe hybridization buffer to 37 ˚ C for 30 min.
3. Remove buffer and pre-hybridize with 400 µL of pre-heated probe hybridization buffer for 30 min at 37 ˚ C.
4. Make probe solution for the target chemosensory receptors (IR8a, IR76b, IR25a, IR41t.1, IR75d, IR7t, IR64a or Orco) by adding 8 pM of probe. Add 8 µL of 1 µM probe stock to 500 µL pre-heated probe hybridization buffer.
5. Remove the pre-heated probe hybridization buffer and replace it with 400 µL of heated probe solution.
6. Incubate tissue at 37 ˚ C for two nights on a nutator placed inside an incubator and covered under a box.

6. Probe amplification

1. Heat up the probe wash buffer to 37 ˚ C. Remove excess probe solution by rinsing the tissue 5x, 10 min per wash, with 400 µL of probe wash buffer at 37 ˚ C, nutating in the incubator.
   NOTE: Preparation for step 6.4 may begin. See the note in step 6.4.
2. Wash samples 2x, 5 min per wash, with 400 µL of 5x saline-sodium citrate containing 10% Tween-20 (SSCT) at room temperature. Thaw amplification buffer to room temperature on a bench top.
3. Prepare tissue for amplification by incubating with 400 µL of amplification buffer for 10 min at room temperature. Due to the viscosity of the amplification buffer, tissues may not be completely submerged. Mix by gently pipetting the amplification buffer below the tissue and expelling the buffer on top of the tissue until they are submerged.
4. Separately prepare 18 pM of hairpin h1 and 18 pM of hairpin h2 by heating 6 µL of 3 µM stock at 95 °C for 90 s and cool to room temperature in a dark drawer for 30 min. Ensure PCR tubes are tightly capped to prevent evaporation of the hairpins while heating in a thermal cycler.
   NOTE: To save time, step 6.4 can be initiated while on the 4^th washing step in step 6.1. Hairpins should be separately heated to prevent cross reaction. Hairpins should not be diluted with any buffer in this step. The hairpins can be purchased from the probe manufacturer along with the probes.
5. Replace the amplification buffer added in step 6.3 with a mixture containing the heated hairpins (from step 6.4) together with 100 µL of amplification buffer. A typical reaction will contain 6 µL of h1, 6 µL of h2, and 100 µL of amplification buffer.
   NOTE: Hairpins h1 and h2 can be combined after cooling to room temperature and then added to 100 µL of amplification buffer. To avoid transferring the tissue samples from one tube to another, the amplification buffer in step 6.3 can be removed and replaced by the hairpins mixture diluted in amplification buffer.
6. Incubate tissue overnight and nutate in the dark at room temperature using the default speed of the nutator.

7. Mounting tissue sample

1. Dilute amplification buffer in the incubated tissue with 300 µL of 5x SSCT (sodium chloride-sodium citrate diluted in Tween-20).
   NOTE: The dilution is helpful to reduce the viscosity and makes it easier to remove the amplification buffer from the tissue. We used Triton-X 100 for the pre-fixative and Tween-20 for the post-fixative step because of the differences in their actions as agents to permeabilize cell membrane.
2. Wash tissue 5x with 400 µL of 5x SSCT at room temperature.
   NOTE: Store tissue temporarily at 4 °C until ready to be mounted, if needed. We have not exceeded 2 days before imaging.
3. Make 5 droplets of mounting solution on a glass slide. Cut the tip of a 200 µL pipette tip to make it wider and transfer the tissue to a new glass slide.
4. Grab the tissue samples by their base with forceps and gently immerse and rinse in a series of mounting solution droplets. Be careful not to break the tissues in this step.
5. Mount with mounting solution, place coverslip, and seal with nail polish. Image in situ tissue samples using a confocal microscope.