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

Our group is interested in the chemosensory system of mosquitoes. We hope that by targeting a mosquito's sense of taste and smell, we can stop it from finding us and biting us. Hybridization chain reaction fluorescent in situ hybridization is a recent development.

This technology is highly sensitive, robust, and capable of multiplexing several gene targets such that we can visualize the expression and localization of these genes simultaneously within their native environment. Special transcriptomics is a cutting edge method used in the field of spatial biology. This technology is capable of multiplexing several thousands of genes, and you can visualize the expression of these genes simultaneously.

Our protocol allows us to directly visualize which neurons in the antenna express any of the 50 or so possible ionotropic receptors. This provides us with a foundational roadmap to guide our understanding of how this class of factor receptors might mediate behaviors. Olfactory neurons within olfactory organs of an insect, like the antenna, are typically buried inside a protective chitinous shell.

This makes visualizing olfactory receptor expression in those neurons very challenging. Our protocol uses a combination of chitinase treatments and a state-of-the-art fluorescence in situ protocol to overcome these challenges. To begin, preheat the dissected Anopheles mosquito heads in CCD buffer at 37 degrees Celsius on a heat block for five minutes.

Transfer the tube with the mosquito heads to a hybridization oven for rotation at 37 degrees Celsius. Next, transfer the entire content of the tube into a dissecting watch glass. With forceps, pick up the heads or any detached antennae and fix them in one milliliter of the pre-fixative.

In a nutator, rotate the heads in the pre-fixative for 24 hours at four degrees Celsius. To perform tissue dissection, first, rinse the heads with one milliliter of 0.1%PBS Tween on ice. Then transfer the heads into a dissecting watch glass.

Under a dissecting microscope, remove tissues of interest from the head with sharp forceps. Hold the anterior part of the head with the forceps, and grab an antenna with another forceps from the base. Similarly, remove the palps, transfer the antennae and palps into empty DNRNase-free tubes placed on ice.

Dehydrate the tissue in 400 microliters of dehydrating reagent for one hour at room temperature. Then replace the dehydrating reagent with 400 microliters of absolute methanol and dehydrate tissues overnight at minus 20 degrees Celsius. After fixation is complete, rehydrate the tissues in a four step series of graded rehydration solutions for 10 minutes on ice.

Then wash the tissues in 400 microliters of PBST for 10 minutes at room temperature. Incubate the tissues in 400 microliters of proteinase K solution for 30 minutes at room temperature. Wash the tissues two times in 400 microliters of PBST to halt the enzymatic digestion.

After the addition of the post fixative, wash the tissue with PBST again before probe hybridization. Submerge the tissue in 400 microliters of probe hybridization buffer for five minutes. Next, remove the buffer and pre-hybridize the tissue in 400 microliters of preheated probe hybridization buffer for 30 minutes at 37 degrees Celsius.

Replace the preheated probe hybridization buffer with 400 microliters of heated probe solution. Place the tube in a nutator for two nights at 37 degrees Celsius. Then place the nutator inside an incubator covered in a box.

Next, nutate the tissue in 400 microliters of probe wash buffer at 37 degrees Celsius in the incubator. After washing the tissues in five times SSCT, incubate in 400 microliters of amplification buffer for 10 minutes at room temperature. Pipet out the amplification buffer from the tube.

Then add a mixture of the heated hairpins H1 and H2.Next, pipet a hundred microliters of amplification buffer. Nutate the tissue overnight in the dark at room temperature. To mount the tissue, first place five drops of mounting solution on a glass slide.

Next, with forceps, grab the washed tissue samples by their base. Gently immerse and rinse in the series of mounting solution droplets. Then mount with mounting solution, and place a cover slip over the tissue.

Seal the cover slip with nail polish before imaging. HCR-FISHed analyses of the female Anopheles mosquito olfactory tissue revealed that IR41t. 1, IR75d, and IR7t were less abundant in the antennae.

IR64a was three times more abundant than the rest of the candidate genes. Colocalization of Orco and IR25a receptor families were observed in the antenna as well as well as in the maxillary palp of the mosquito. Colocalization patterns suggest that IR76b positive cells express IR25a, whereas IR8a positive cells partially colocalize with IR76b and IR25a.