Die Autoren danken Vasia Vagin für die Einführung dieser Technik in unserem Labor.

<ol><li> Machen Sie einen Glasobjektträger mit einem schwarzen Hintergrund: Cut 20 # weißes Papier auf die gleiche Größe wie die Objektträger. Farbe der einen Seite der Folie-Papier schwarz mit einem Permanent-Marker (zB Sharpie). Bewerben Weißleim (Elmer), die schwarze Seite und halten das Papier auf die Folie. Die Folie kann wiederverwendet werden. Mit ¾ &quot;Etikettenband (Fisherbrand), sichern Sie die Objektträger, Glas nach oben, um die CO 2-fly-Pad, um es zu fixieren. </li><li> Sammeln Sie 0-3 Tage alten männlichen Fliegen. </li><li> Bereiten Sie einen Eimer mit Eis, Kochsalzlösung (PBS), seziert Nadeln phosphatgepuffertes. </li><li> Betäuben 50-100 fliegt mit einem Strom von CO 2 und übertragen Sie sie aus der Flasche oder Fläschchen-the-fly-Pad in der Nähe der Objektträger. </li><li> Pipette ~ 50 ul eiskaltem PBS in einem Tropfen in der Mitte des Objektträgers. </li><li> Gently abholen eine männliche Fliege mit dem Präpariernadel-dip die Präpariernadel in PBS, sanfte Berührung der Bauchseite (dh in der Nähe der Flügel) der Fliege mit der Nadel-Platz in die Tropfen PBS. </li><li> Legen Sie eine Nadel auf die Mitte des zu fliegen, zwischen den Brustkorb und den Bauch, drücken Sie fest. Legen Sie die andere Nadel zwischen dem sechsten (A6) und siebten (A7) Abdominalsegmente (die zweite und dritte Streifen von unten); ziehen Sie den Bauch vom Brustkorb. </li><li> Tease den Hoden von anderen Organen, vor allem im Darm. Besonders in weißen Mutanten, schauen Hoden und Darm ähnlich. Wesentliche Unterschiede sind, dass Hoden immer in Paaren, jeder Hoden hat eine spiralförmige Muster und ist in der Farbe weiß, als gut. </li><li> Bewahren Sie die Hoden in eiskaltem PBS in einem Polypropylen-Mikrozentrifugenröhrchen. </li></ol> Repräsentative Ergebnisse: <img src="/files/ftp_upload/2641/2641fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Dorsale Ansicht der männlichen Geschlechtsorgane, von flybase.org/reports/FBim0000074.html. FlyBase Zuordnung:... &quot;Nach Abbildung 38 A auf Seite 508 aus Miller (1950) A. Miller Die innere Anatomie und Histologie der imago von Drosophila melanogaster In M. Demerec, Redakteur, Biologie von Drosophila, Kapitel 6, Seiten 420 - 534. Cold Spring Harbor Laboratory Press, Faksimile-Ausgabe, 1994. Copyright 1994 von Cold Spring Harbor Laboratory Press. mit freundlicher Genehmigung von Cold Spring Harbor Laboratory (CSHL) Drücken verwendet. Siehe <a href="http://www.cshlpress.com">http://www.cshlpress.com</a> . &quot;

<ol>
	<li>Singh, S. R., Hou, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistological+Techniques+for+Studying+the+Drosophila+Male+Germline+Stem+Cell.">Immunohistological Techniques for Studying the Drosophila Male Germline Stem Cell.</a> Methods Mol Biol. 450, 45-59 (2008).</li></ol>

Wir bieten ein Protokoll zum Hoden von ausgewachsenen männlichen Drosophila melanogaster zu isolieren. Da die Technik erfordert Koordination eigenen zwei Hände, Geschick geht nur mit der Praxis.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dissecting needle</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>S17346</td>
<td>&nbsp;</td>
<tr>
<td>Micro Slides, Plain </td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>12-553</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Light</td>
<td>Tool</td>
<td>Schott-Fostec</td>
<td>KO-1500</td>
<td>&nbsp;</td>
<tr>
<td>CO2 pad</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>59-172</td>
<td>&nbsp;</td>
<tr>
<td>Benchtop Flowbuddy Complete System</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>59-122WCU</td>
<td>&nbsp;</td>
<tr>
<td>Bubble Humidifier</td>
<td>Tool</td>
<td>Salter Labs</td>
<td>7100</td>
<td>&nbsp;</td>
<tr>
<td>Ball filler needle</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>54-119</td>
<td>&nbsp;</td>
<tr>
<td>&frac34;&#x201D; Label Tape</td>
<td>Supply</td>
<td>Fisher Scientific</td>
<td>15-901-5B</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of drosophila melanogaster testes

Die Hoden von Drosophila melanogaster stellen ein wichtiges Modell für das Studium der Erhaltung von Stammzellen und Differenzierung, Meiose, und Soma-Keimbahn-Wechselwirkungen. Testes sind in der Regel von männlichen Erwachsenen 0-3 Tage nach dem Schlüpfen aus der Puppenhülle isoliert. Die Hoden von Wildtyp-Fliegen sind leicht von anderen Geweben zu unterscheiden, weil sie gelb sind, aber die Hoden aus weißem mutierte Fliegen, sind eine häufige genetische Hintergrund für Laborexperimente ähnlich in Form und Farbe, um die Fliege gut. Performing Dissektion auf einem Glasobjektträger mit einem schwarzen Hintergrund macht die Identifizierung der Hoden erheblich erleichtert. Hoden werden von den Fliegen mit Sezieren Nadeln entfernt. Im Vergleich zu Protokollen, die Zange verwenden für Hoden Dissektion, ist unsere Methode viel schneller, so dass ein eingespieltes Einzelnen Hoden 200 bis 300 Wildtyp-Fliegen pro Stunde zerlegen, wodurch 400-600 Hoden. Testes von weißen Fliegen oder von Mutanten, die Hoden verkleinern, sind schwerer zu sezieren und liefern typischerweise 200-400 Hoden pro Stunde.

Watch this Scientific Journal Video about Isolation of Drosophila melanogaster Testes at JoVE.com

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster Hoden können schnell und effizient von erwachsenen Männern mit Sezieren Nadeln isoliert. Mit etwas Übung kann man leicht in ein oder zwei Tage zu isolieren einen Betrag von Hoden reicht für die Analyse von DNA oder RNA, die im Hochdurchsatz-Sequenzierung oder eher traditionellen Methoden der Molekularbiologie oder der Protein-Antikörper-oder Enzym-Assays.

Isolierung von 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 ...

isolation-of-drosophila-melanogaster-testes

Research

JoVE Journal

Biology

Isolation of Drosophila melanogaster Testes

32.3K Aufrufe.  University of Massachusetts Medical School. Drosophila melanogaster Hoden können schnell und effizient von erwachsenen Männern mit Sezieren Nadeln isoliert. Mit etwas Übung kann man leicht in ein oder zwei Tage zu isolieren einen Betrag von Hoden reicht für die Analyse von DNA oder RNA, die im Hochdurchsatz-Sequenzierung oder eher traditionellen Methoden der Molekularbiologie oder der Protein-Antikörper-oder Enzym-Assays.

Serie: Isolation of Drosophila melanogaster Testes

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.

Dissection der Larven ZNS von 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 ...

Forschung

Biologie

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.

Hochauflösendes Video Tracking von 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 ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Live-Bildgebung Drosophila melanogaster Embryonale Hemocyte Migrationen

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

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.

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.

Präparation Oenocytes von Adult Drosophila melanogaster</em

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

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Messung der Lebensdauer in<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Die zytologische Analyse der Spermatogenese: Live-und Fest Zubereitungen aus<em&gt; Drosophila</em&gt; Hoden

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging Durch die Puppenhülle von<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

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.

Präparation und Immunfärbung von Imaginalscheiben aus<em&gt; Drosophila melanogaster</em

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

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Verbesserte Northern Blot Detektion von kleinen RNA-Spezies in<em&gt; Drosophila melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Aufrechterhaltung eines<em&gt; Drosophila melanogaster</em&gt; Bevölkerung Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...