一个简单的一步解剖协议成人全安装准备

The overall goal of this protocol is to describe a simple one step dissection procedure for adult Drosophila brains, which can quickly produce brains with well preserved morphology suitable for whole mount analysis. This method can help answer questions in the fields of neuroscience and genetics. This includes fine mapping of brain circuitries, studying the effects of genetic manipulations of neurons, and functional characterization of genes.

The main advantage of this technique is that it involves an easy to learn procedure. It can facilitate in dissecting out an adult fly brain with well preserved morphology in less than 10 seconds. Video demonstration of this method is critical, as controlling the required momentum when releasing the forceps to tear away the exoskeleton surrounding the fly head takes skills and practice.

These tiny, annoying fruit flies have taught us a lot about the biological principles. They may also help us find the causes and the cures for human diseases, such as Alzheimer's disease, and Parkinson's disease. Using a dissecting microscope set to 1.2 X magnification, immobilize an anesthetized fly and immerse it into cold dissection solution with the abdomen facing upwards.

Keep the forceps at a 160 to 170 degree angle respective to the dissection plate, and then exert a small force onto the abdomen of the fly. This should immobilize the fly, force the head backwards 15 to 25 degrees, and extend the proboscis upwards. After this maneuver, a soft translucent region of the cuticle underneath the proboscis should become apparent.

Increase the magnification and adjust the focal plane onto this region. Using the dominant hand, take a pair of dissecting forceps and exert pressure to close the tips. Position the forceps perpendicularly to the forceps restraining the fly.

Pierce the closed tips of the forceps through the soft translucent region of the cuticle, taking care not to penetrate so deeply that they touch the brain. Then quickly, but steadily, release the forceps, so that the points open, tearing away the exoskeleton of the fly head. Use the momentum from releasing the forceps tips to gently remove the exoskeleton and most of the head case associated eye and trachea from the brain tissue in one motion.

An intact brain should now be visible. Using the forceps, carefully remove the remaining accessory tissues, such as the white fibrous tracheal tissue, taking care not to pull or damage the brain proper. Place the dissected brain samples with associated torsos into approximately one milliliter of 4%paraformaldehyde on ice for 40 to 60 minutes.

Remove the paraformaldehyde, and then rinse the samples with one milliliter of one X PBS by pipetting the solution up and down three times, taking care not to aspirate the brains. Next, remove the PBS and then wash five times with one milliliter of one X PBT for five minutes each with nutation. Add the primary antibody of interest at the appropriate dilution.

And then incubate the samples overnight at four degrees Celsius with nutation. Remove the primary antibody and wash the samples five times with one milliliter of one X PBT for five to 10 minutes each wash with nutation. Add secondary antibody to the washed samples at the appropriate dilution and incubation time.

Following this, wash the samples six times in one milliliter of one X PBT for 10 minutes each with nutation. This step is critical to remove any non-specifically bound secondary antibodies. Incubate the samples in a one to 10, 000 dilution of one milligram per milliliter DAPI in one milliliter of one X PBT solution for 30 to 60 minutes with nutation.

Finally, rinse the samples by adding one milliliter of one X PBS and then pipetting the solution up and down three times. Place a cover slip onto a mounting slide. Using a pipette with a cut tip, transfer the brains in one X PBS solution onto the coverslip.

Next, using forceps, separate the brains from the bodies. Use a fine pipette tip to remove the liquid around the brain tissue. And then, add 20 to 40 microliters of anti-fade mounting medium.

Gently spread the brains, whilst displacing the viscous medium outwards. Beginning at one side, gently lower a second coverslip onto the samples, taking care to minimize brain contact with bubbles. Allow the slips to settle, and then remove any excess liquid from the edge of the slips using a laboratory wipe.

Use a small piece of tape to temporarily attach the coverslip pair to the mounting slide. To image the samples, use a compound fluorescent or confocal microscope. These images show anterior and posterior views of the same adult Drosophila.

Cell nuclei were visualized by DAPI staining and neurons were labeled using a pan neuronal marker and anti-ELAV antibodies. Dopaminergic, or DA, neurons were revealed by anti-TH antibody stain. Similar levels of intensity were seen between both sides of the brain for all image channels.

Enlarged views of the anterior and posterior of the brain allow detailed examination of the DA neuronal clusters. The paired anterior medial cluster can be seen in the anterior images, and the paired posterior medial and paired posterior lateral clusters in the posterior side of the brain. Quantification of these neurons revealed that three day old male w1118 strain flies typically have 27 DA neurons in the PPM cluster in the whole brain, 16 in the PPL cluster per hemisphere, and five in the PAL cluster per hemisphere.

An average of 97 DA neurons was seen in each of the PAM clusters. 3D reconstruction of the PAL and PAM clusters also showed that the DA neurons in the PAM cluster have relatively small sizes and weaker TH staining than those in the other clusters, such as the PAL. Increasingly, more studies are focusing on adult fly brains for dissecting molecular pathways and neuronal circuitry underlying higher brain functions, and for modeling human brain diseases.

In such brain based studies it will be necessary to efficiently prepare brain samples with well preserved morphology. This could be especially important in large scale brain based screens. I first had the idea for this method when struggling to hold flies in the forceps.

Instead of forceps with sharp tips that most Drosophila researchers use, Shebna tried blunt end forceps and found that it was easier to dissect the brains. Once mastered, this technique can be done in less than 10 seconds if it is performed properly. Once, I dissected about 100 brains of six different genotype for one experiment in just one day.

While this dissection procedure may look simple, it's important to practice it first with dispensable flies. The investigator may also struggle with positioning the forceps without destroying the fly brain. The dissected brains can be used for other studies, like RNA, in situ hybridization, and extraction of protein and RNA sample from brain tissue.

After watching this video, you should be able to dissect fly brains with well preserved morphology. We predict that improvements can be developed to make this procedure simpler and faster. This can potentially become automated in the future for large scale studies.

Don't forget that working with the paraformaldehyde and the sharp dissecting forceps can be hazardous. Take precautions, such as wearing gloves, when handling fixative solutions, and once the dissection is complete immediately cap the forceps before putting them aside.