We acknowledge Mr. Enes Mehmet, Ms. Kiara Andrade, Ms. Pilar Rodriguez, Chris Kwok, and Ms. Danna Ghafir for their immense support to the project.

1. Solutions Used for Brain Dissection and Immunofluorescent Staining
<ol>
	<li>Dissect the adult fly brains in artificial Cerebral Spinal Fluid (aCSF): 119 mM NaCl, 26.2 mM NaHCO3, 2.5 mM KCl, 1 mM NaH2PO4, 1.3 mM MgCl2, and 10 mM glucose. Before use, gas the aCSF with 5% CO2/95% O2 for 10 - 15 min and spike with 2.5 mM CaCl2. Sterilize the aCSF solution by filtering through a 0.22 &#181;m membrane filter. 
	Note: Store ...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+tools+for+multicolor+stochastic+labeling+reveal+diverse+stereotyped+cell+arrangements+in+the+fly+visual+system.">Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system.</a> Proc Natl Acad Sci U S A. 112, 2967-2976 (2015).</li><li>Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Plasticity:+Dopamine+Tunes+the+Mushroom+Body+Output+Network.">Neural Plasticity: Dopamine Tunes the Mushroom Body Output Network.</a> Curr Biol. 26, 109-112 (2016).</li><li>Wolff, T., Iyer, N. A., Rubin, G. 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S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+dissecting+Drosophila+melanogaster+brains+for+live+imaging+or+immunostaining.">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat Protoc. 1, 2110-2115 (2006).</li><li>Mao, Z., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eight+different+types+of+dopaminergic+neurons+innervate+the+Drosophila+mushroom+body+neuropil:+anatomical+and+physiological+heterogeneity.">Eight different types of dopaminergic neurons innervate the Drosophila mushroom body neuropil: anatomical and physiological heterogeneity.</a> Front Neural Circuits. 3, 5 (2009).</li><li>White, K. E., Humphrey, D. M., Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopaminergic+system+in+the+aging+brain+of+Drosophila.">The dopaminergic system in the aging brain of Drosophila.</a> Front Neurosci. 4, 205 (2010).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathology+and+muscle+and+dopaminergic+neuron+degeneration+caused+by+inactivation+of+Drosophila+Pink1+is+rescued+by+Parkin.">Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin.</a> Proc Natl Acad Sci U S A. 103, 10793-10798 (2006).</li><li>Greene, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathology+and+apoptotic+muscle+degeneration+in+Drosophila+parkin+mutants.">Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants.</a> Proc Natl Acad Sci U S A. 100, 4078-4083 (2003).</li><li>Whitworth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+glutathione+S-transferase+activity+rescues+dopaminergic+neuron+loss+in+a+Drosophila+model+of+Parkinson's+disease.">Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease.</a> Proc Natl Acad Sci U S A. 102, 8024-8029 (2005).</li><li>Pesah, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+parkin+mutants+have+decreased+mass+and+cell+size+and+increased+sensitivity+to+oxygen+radical+stress.">Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress.</a> Development. 131, 2183-2194 (2004).</li><li>Trinh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decaffeinated+coffee+and+nicotine-free+tobacco+provide+neuroprotection+in+Drosophila+models+of+Parkinson's+disease+through+an+NRF2-dependent+mechanism.">Decaffeinated coffee and nicotine-free tobacco provide neuroprotection in Drosophila models of Parkinson's disease through an NRF2-dependent mechanism.</a> J Neurosci. 30, 5525-5532 (2010).</li><li>Kim, K., Kim, S. H., Kim, J., Kim, H., Yim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+s-transferase+omega+1+activity+is+sufficient+to+suppress+neurodegeneration+in+a+Drosophila+model+of+Parkinson+disease.">Glutathione s-transferase omega 1 activity is sufficient to suppress neurodegeneration in a Drosophila model of Parkinson disease.</a> J Biol Chem. 287, 6628-6641 (2012).</li></ol>

With an increasing interest in using adult Drosophila brain to study human brain diseases, neuronal circuitry, and higher brain functions, it is necessary to develop simple and quick methods to obtain intact fly brains for whole-mount analyses, which is especially important for large-scale brain-based screens. Our method provides a simple and easy-to-learn approach to dissect out a fly head (often in less than 10 s&#160;with experience) with well-preserved morphology that is largely cleared of associated tissues...

The model organism Drosophila, commonly known as the fruit fly, has long been valued for its elegant genetic tools, short reproductive times, and highly conserved molecular and cellular pathways. The fruit fly has been successfully employed to dissect basic signaling pathways, the patterning mechanisms of multicellular organisms, as well as the mechanisms underlying neuronal development, functions, and diseases1,2. With recent advances in cell labeling and imaging technologies, the fruit fly brain has become especially powerful in fine mapping of neuronal circuitry and in dissecting the molecular and cellular basis of higher brain functions, such as learning and memory, and circadian rhythm1,3,4,5,6,7,8.
One particular advantage of the Drosophila system is its relatively small size, allowing whole-mount preparation and examination of the brain using a regular compound or confocal microscope. This feature enables detailed anatomic and functional analyses of neuronal circuitry, or even a single neuron, at cellular and subcellular levels, in the context of a whole brain tissue, thus providing both a holistic view of the studied subject and its exact geometry within the whole brain. However, given the rather miniature size of the brain, it also presents a technical challenge in efficiently dissecting an intact brain tissue out of the protective exoskeleton head case in an adult fly. Various effective and relatively simple dissection methods have been described in detail, which usually involve careful and step-wise removal of the head case and the associated tissues including the eyes, trachea, and fat from the brain proper9, 10. These microsurgical dissection methods often place rather stringent demands on the quality of the dissection forceps, relying on forceps with fine well-aligned tips that can be easily damaged. Moreover, as the dissected brains are often separated from the rest of the body, the brains can be easily lost during the subsequent staining and washing processes because of their small sizes and their transparency in the processing buffer. Here, we describe a relatively simple and easy-to-learn, one-step dissection protocol for adult brains that keeps the dissected brains attached to the torso. The dissection process often easily clears away most of the brain-associated tissues such as the eye and trachea and reduces the demand for good quality dissection forceps.
Additionally, when imaging the brain under the fluorescent compound microscope or confocal microscope, the side of the brain that is away from the fluorescent light source often produces a weaker signal and less clear images due to the thickness of the whole-mount brain. Here, we also describe a simple mounting method that allows easy flipping of the brain samples, enabling convenient imaging of both sides of the brain with similar signal intensity and quality.
As a proof-of-concept for the application of this method to study the adult brain, we further examined the presence of DA neurons in the brains of w1118 flies; a genotype that is often used as the parental line for generating transgenic flies and the wildtype control in many Drosophila studies.

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			<td height="54" style="height:54px;width:573px;">w*;&#160;park&#916;21/TM3,&#160;P{GAL4-Kr.C}DC2,&#160;P{UAS-GFP.S65T}DC10,&#160;Sb1</td>
			<td style="width:311px;">Bloomington Drosophila Stock Center</td>
			<td style="width:151px;">51652</td>
			<td style="width:287px;">Balancer was switched to TM6B</td>
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			<td height="39" style="height:39px;">PBac{WH}parkf01950</td>
			<td>Exelixis at Harvard Medical School</td>
			<td>f01950</td>
			<td>Balancer was switched to TM6C</td>
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			<td style="width:311px;">Fisher Scientific</td>
			<td>S640-500</td>
			<td></td>
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			<td height="25" style="height:25px;width:573px;">Sodium Bicarbonate (NaHCO3)&#160;</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td style="width:151px;">02-003-990</td>
			<td></td>
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			<td height="21" style="height:21px;width:573px;">Potassium Chloride (KCl)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
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			<td height="25" style="height:25px;width:573px;">Sodium phosphate, monobasic monohydrate (NaHCO3)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-004-198</td>
			<td></td>
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			<td height="21" style="height:21px;width:573px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-003-265</td>
			<td></td>
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			<td height="21" style="height:21px;width:573px;">D-Sorbitol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>S1876-500G</td>
			<td>Replaces glucose</td>
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			<td height="25" style="height:25px;width:573px;">Calcium chloride dihydrate (CaCl2)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>C5670-500G</td>
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			<td height="21" style="height:21px;">EMD Millipore&#160;Durapore PVDF Membrane Filters: Hydrophilic: 0.22&#181; Pore Size</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>GVWP14250</td>
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			<td height="21" style="height:21px;width:573px;">Formalin Solution, 10% (Histological)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>SF98-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Potassium Phosphate, Dibasic, Powder, Ultrapure Bioreagent</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-003-823</td>
			<td></td>
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			<td height="21" style="height:21px;width:573px;">Tween 20</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Excelta Precision Tweezers with Very Fine Points</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>17-456-055</td>
			<td>Protocol does not require very fine points.&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Anti-Tyrosine Hydroxylase Antibody</td>
			<td>Pel-Freez Biologicals</td>
			<td style="width:151px;">P40101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Rat-Elav-7E8A10 anti-elav</td>
			<td>The Developmental Studies Hybridoma Bank</td>
			<td>Clone 7E8A10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">A-21247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">A-11037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">DAPI Solution (1 mg/mL)</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">62248</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Propyl gallate powder</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>P3130-100G</td>
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			<td height="21" style="height:21px;width:573px;">Glycerol ACS reagent, &#8805;99.5%</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>G7893-500ML</td>
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			<td height="21" style="height:21px;">Zeiss Axioimager Z1</td>
			<td style="width:311px;">Zeiss</td>
			<td style="width:151px;">Quote</td>
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			<td height="21" style="height:21px;width:573px;">Zeiss Apotome.2</td>
			<td style="width:311px;">Zeiss</td>
			<td style="width:151px;">Quote</td>
			<td></td>
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			<td height="21" style="height:21px;width:573px;">Zen lite software</td>
			<td style="width:311px;"></td>
			<td style="width:151px;">Quote</td>
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a simple one-step dissection protocol for whole-mount preparation of adult drosophila brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

Figure 1 illustrates the main procedures for adult brain dissection, as described above. Figures 2 and 3 are representative images of 3-day-old WT (genotype: w1118) adult fly brains, which were costained with an antibody against Tyrosine Hydroxylase (TH, colored in red in Figure 2 and white in Figure 3), a marker commonly used to label DA neurons11, in addition to the DNA dy...

Watch this Scientific Journal Video about A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains at JoVE.com

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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.

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26.7K visualizações.  The University of Texas Health Science Center at Houston. 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...