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 DNA/RNA 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 re-hydration 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 pre-heated probe hybridization buffer for 30 minutes at 37 degrees Celsius. Replace the pre-heated 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 5x SSCT, incubate in 400 microliters of amplification buffer for 10 minutes at room temperature. Pipette out the amplification buffer from the tube. Then add a mixture of the heated hairpins, H1 and H2.Next, pipette 100 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 fiche analyses of the female anopheles mosquito olfactory tissue revealed that IR 41t1, IR75d, and IR7t were less abundant in the antennae. IR64a was three times more abundant than the rest of the candidate genes. Co-localization of orco and IR25a receptor families were observed in the antenna as well as in the maxillary palp of the mosquito.
Co-localization patterns suggest that IR76b positive cells express IR25a, whereas IR8a positive cells partially co-localize with IR76b and IR25a.
