We would like to thank Amsale Belay for her assistance in filming of this video article. This work was funded by CIHR, NSERC and CRC grants to JDL.

Part 1. Fillet preparation of the adult Drosophila abdomen.
The procedure outlined immediate below prepares the abdomen for the subsequent removal of the oenocytes (see Part2 of protocol). The same procedure can also be used for the preparation of the other tissues attached to the inner surface of the cuticle, including the dorsal vessel (i.e. heart) and fat body.
Note that in the video article the oenocyte dissection technique is demonstrated on an adult wild-type (Canton-S) male fly, six days of age. Identical techniques can be used to remove the oenocytes from female flies. The abdomen fillet preparation takes approximately 5 minutes to perform.
Before beginning it is important to have ready several fine dissection pins (see below and Part 2) and a dissection needle (see Part 2). Commercially available dissection pins and needles are often too large for this protocol, so we have taken to fabricating our own in lab. To create the dissection needle, tungsten wire (0.005") is first threaded through the shaft of hypodermic needle (27G, aluminum hub). Thread the wire through the tip of the hypodermic needle so that it emerges from the hub. Crimp the wire protruding from hub; this prevents the wire from being pulled out of the hypodermic needle. Next, cut the wire at the opposite end of the crimp such that several millimeters (~3-4mm) of wire protrude from the tip of the hypodermic needle. Passing the tip of exposed tungsten through a flame will sharpen the wire to a very fine point. A disposable 5ml plastic pipette when wedged into the hub of the hypodermic needle acts as convenient handle for this dissection tool. The fine dissection pins are made in a similar way. After the sharpening step, grasp the very tip of the wire firmly with forceps to prevent it from moving and make a cut approximately 3mm from the tip to release the small dissection pin. Use care to prevent pin from being flung from the grasp of the forceps.
To fillet prepare the adult Drosophila abdomen:
<ol>
 <li> Secure the fly to the dissection plate (Sylgard; Dow-Corning) by placing a fine dissection pin though the thorax.</li>
 <li> Remove the legs and wings of the fly. While removing the most posterior set of legs, create an opening in the cuticle at the point just anterior to where the thorax meets the abdomen. This opening is used later as an access point for making an incision along the ventral surface of the abdomen (see Step 5)</li>
 <li> Secure the abdomen of the fly in place by placing a second dissection pin through the genital segment.</li>
 <li> Cover the fly with chilled Shields and Sang M3 insect medium (Sigma). Remove any trapped air by drawing it off with a Pasteur pipette.</li>
 <li> Using forceps open the abdomen by making an incision along the ventral midline from the opening in the cuticle created by removing the legs to the genital segment. Remove the guts and gonads from the abdomen.</li>
 <li> Sever the cuticle and tracheae connecting the abdomen to the thorax. The only remaining tissue connecting the abdomen to the thorax should be the heart. This provides the abdominal cuticle with some stability, and prevents it from rotating or twisting during the next step.</li>
 <li> Pin flat the anterior most corners of the abdominal cuticle. Remove the thorax. Pulling the cuticle taut, pin flat the posterior corners of the cuticle. The pin in the genital segment may need to be repositioned to make the cuticle lay flat.</li>
</ol>
Part 2. Oenocyte dissection
The abdomen fillet preparation exposes the internal surface of the abdominal cuticle and attached tissues including the heart, fat body, tracheae, body wall muscles, epidermis, and the oenocytes. The heart lies along the dorsal midline. Fat body (opaque tissue) covers most of the internal cuticular surface. Tracheae (white tubular structures) extend from the lateral spiracle openings, and make extensive branches which infiltrate these tissues. Lying beneath these tissues, the pigmented oenocytes (amber-colored cells) radiate from the midline to the lateral edge of the cuticle in the posterior region of each segment. The oenocytes lie directly beneath the darkly tanned regions of each tergite (dorsal cuticular plates) and can be difficult to identify without prior knowledge of their location. Another population of ventrally located oenocytes is associated with the sternites (ventral cuticular plates); although it is possible to isolate these cells, this video protocol will only demonstrate the dissection of the dorsal oenocytes. In each abdominal segment, a prominent leaf of fat body tissue lies anterior to the oenocytes; a smaller less obvious leaf of fat body lies posterior to the oenocytes. The prominent leaf of fat body tissue often covers the oenocytes of the segment preceding it. The dorsal vessel and fat body must be removed first in order to expose the oenocytes.
It is important to note that the amber pigmentation of the oenocytes, from which the their name derives (oeno- from Greek o&icirc;nos, 'wine')1, clearly distinguishes these cells from the surrounding tissues; the intensity of the pigmentation increases with the age of the fly. Moreover, a basal lamina ensheathes the oenocytes2,3, providing support to the cell clusters and preventing the cells from becoming dissociated during the dissection. The basal lamina also aids in the removal of extraneously adhered tissues, such as fat body tissue, by creating a weak point where these adhesions can be easily disrupted.
The procedure outlined below provides detailed instructions for the removal of the oenocytes from the abdominal integument. The oenocytes can be easily removed from abdominal segments 2-5 (male), and 2-6 (female). The oenocytes from the first and last abdominal segments (segments 1, and 6 in males or 7 in females) are often disrupted by the dissection pins holding the corners of the cuticle and are therefore avoided. The removal of the oenocytes takes approximately 10 minutes to perform.
To isolate the adult oenocytes:
<ol>
 <li> Sever the tracheal trunks emanating from the spiracles using a dissection needle.</li>
 <li> Remove the fat body and heart by severing the connections to the inner surface of the cuticle with a dissection needle. To do so, position the point of the dissection needle between the fat body and the oenocytes, and drag it from the lateral edge of the cuticle, through to the midline, moving underneath the heart, and continuing to opposite side. Progressing from the posterior to anterior, remove the fat body tissue and the heart from the cuticle of each successive segment. If performed carefully the oenocytes remain attached to the cuticle, and the fat body and heart can be removed whole.</li>
 <li> Body wall muscles strap the oenocytes to the inner surface of the cuticle. Using the dissection needle sever the longitudinal body wall muscles of each segment. To do so, drag the dissection needle along the posterior side of the oenocyte strand, gently lifting them from the surface of the cuticle. The ribbon-like oenocyte strands should come away from the cuticle and can be laid to the side while the remaining oenocytes are removed.</li>
 <li> Once removed from the cuticle and lying on the dissection plate it may be necessary to clean off any adhered to fat body tissue from the oenocytes. This is done by simply placing the dissection needle between the fat body tissue and the oenocytes, and severing the connection by pushing the needle down and against the dissection plate.</li>
 <li> Gather the oenocyte strands by simply poking them with the end of the dissection needle. Once stuck to the end of the dissection needle the oenocytes can be pulled slowly across the surface of the culture medium. The dissected oenocytes can be placed directly into cell lysis buffer appropriate for either RNA or protein extraction. Note that the oenocytes (abdominal segments 2-5) from a single male fly typically yields 15-20ng of total RNA.</li>
</ol>
Figure Legend:
<img src="/files/ftp_upload/2242/2242fig1.jpg" alt="Figure 1" /> 
 Figure 1. Expression of the desaturase genes, desat1 and desatF, in male and female oenocytes of D. melanogaster. Reverse transcription-PCR amplification of desat1 and desatF from cDNA derived from adult male and female oenocytes. The desat1 gene is expressed in both male and female oenocytes; desatF expression is sexually dimorphic, being expressed exclusively in female oenocytes. Corresponding to these differences in gene expression, desat1 is required for the production of hydrocarbon compounds common to both males and females9, and desatF is involved in the production of female-specific hydrocarbon compounds10,11. desatF has been shown to be specifically expressed in female oenocytes by in situ hybridization12. Note that on average 15-20ng of total RNA is obtained from the oenocytes dissected from an individual fly. RNA was isolated using the RNeasy micro kit (Qiagen); cDNA was produced using the qScript cDND synthesis kit (Quanta BioSciences)

<ol>
	<li>Wielowiejski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ueber+das+blutgewebe+der+insekten.">Ueber das blutgewebe der insekten.</a> Zeit. Wiss. Zool. 43, 512-536 .</li><li>Krupp, J. J., Kent, C., Billeter, J. C., Azanchi, R., So, A. K., Schonfeld, J. A., Smith, B. P., Lucas, C., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+experience+modifies+pheromone+expression+and+mating+behavior+in+male+Drosophila+melanogaster.">Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster.</a> Curr. Biol. 18, 1373-1383 (2008).</li><li>Locke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+membranes,+Golgi+complexes,+and+vacuolar+systems.">Surface membranes, Golgi complexes, and vacuolar systems.</a> Annu. Rev. Entomol. 48, 1-27 (2003).</li><li>Billeter, J. C., Atallah, J., Krupp, J. J., Millar, J. G., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+cells+tag+sexual+and+species+identity+in+Drosophila+melanogaster.">Specialized cells tag sexual and species identity in Drosophila melanogaster.</a> Nature. 461, 987-991 (2009).</li><li>Gould, A. P., Elstob, P. R., Brodu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+oenocytes:+a+model+system+for+studying+cell-fate+specification+by+Hox+genes.">Insect oenocytes: a model system for studying cell-fate specification by Hox genes.</a> J. Anat. 199, 25-33 (2001).</li><li>Lawrence, P. A., Johnston, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+the+Drosophila+abdomen:+the+epidermis,+oenocytes+and+ventral+muscles.">Cell lineage of the Drosophila abdomen: the epidermis, oenocytes and ventral muscles.</a> J. Embryol. Exp. Morphol. 72, 197-208 (1982).</li><li>Johnson, M. B., Butterworth, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+and+aging+of+adult+fat+body+and+oenocytes+in+Drosophila+as+revealed+by+light+microscopic+morphometry.">Maturation and aging of adult fat body and oenocytes in Drosophila as revealed by light microscopic morphometry.</a> J. Morphol. 184, 51-59 (1985).</li><li>Gutierrez, E., Wiggins, D., Fielding, B., Gould, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+hepatocyte-like+cells+regulate+Drosophila+lipid+metabolism.">Specialized hepatocyte-like cells regulate Drosophila lipid metabolism.</a> Nature. 445, 275-280 (2007).</li><li>Marcillac, F., Bousquet, F., Alabouvette, J., Savarit, F., Ferveur, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+with+major+effects+on+Drosophila+melanogaster+sex+pheromones.">A mutation with major effects on Drosophila melanogaster sex pheromones.</a> Genetics. 171, 1617-1628 (2005).</li><li>Chertemps, T., Duportets, L., Labeur, C., Ueyama, M., Wicker-Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+female-specific+desaturase+gene+responsible+for+diene+hydrocarbon+biosynthesis+and+courtship+behaviour+in+Drosophila+melanogaster.">A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster.</a> Insect Mol. Biol. 15, 465-473 (2006).</li><li>Legendre, A., Miao, X. X., Da Lage, J. L., Wicker-Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+a+desaturase+involved+in+female+pheromonal+cuticular+hydrocarbon+biosynthesis+and+courtship+behavior+in+Drosophila.">Evolution of a desaturase involved in female pheromonal cuticular hydrocarbon biosynthesis and courtship behavior in Drosophila.</a> Insect. Biochem. Mol. Biol. 38, 244-255 (2008).</li><li>Shirangi, T. R., Dufour, H. D., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+sex+pheromone-producing+enzyme+expression+in+Drosophila.">Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.</a> PLoS Biol. 7, e1000168-e1000168 (2009).</li></ol>

No conflicts of interest declared.

In this video article we present a detailed dissection protocol for the preparation of the oenocytes from adult D. melanogaster in a manner suitable for molecular analysis. An abbreviated text-based account of the dissection method has been described elsewhere2, and the resulting oenocyte preparation has been demonstrated to be appropriate for the extraction of both RNA and protein2. Using this preparation it may also be possible to develop methods for the culturing of explanted oenocytes....

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		<th>Comment</th>
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<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tungsten Wire</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>27-11</td>
<td>0.005&#x201D;/20&#x2019;</td>
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<td>Forceps</td>
<td>&nbsp;</td>
<td>Dumont</td>
<td>11252-30</td>
<td>#5</td>
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<td>Shields and Sang M3 Insect Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
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<td>RNeasy Micro Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>74004</td>
<td>&nbsp;</td>
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<td>qScript cDNA Synthesis Kit</td>
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<td>Quanta Biosciences</td>
<td>95047-100</td>
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dissection of oenocytes from adult drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Watch this Scientific Journal Video about Dissection of Oenocytes from Adult Drosophila melanogaster at JoVE.com

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

dissection-of-oenocytes-from-adult-drosophila-melanogaster

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20.4K visualizações.  University of Toronto. Em insetos, o oenocytes produzir compostos de hidrocarbonetos cuticulares. Estes compostos protegem contra a dessecação e facilitar a comunicação química. Aqui nós demonstramos uma técnica de dissecção usado para isolar o oenocytes de adultos Drosophila melanogaster, E ilustram como esta preparação pode ser utilizada para estudar os genes envolvidos na síntese de hidrocarbonetos.

Vídeo: Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Drosophila Ovaries

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...