Gli autori desiderano ringraziare Akira Chiba per il sostegno intellettuale, supporto materiale, e le scorte. Grazie a Julia Dallman per i commenti.

1. Vola lavoro <ol><li> Mantenere mosche sul medio farina di mais-agar-melassa, lievito normale a temperatura ambiente 10. </li><li> Per croci, isolare vergini entro 6 ore di eclosion. Dopo aver attraversato ai maschi del genotipo desiderato, il cambiamento vola a nuovi flaconi ogni 3-4 giorni. </li></ol> Nota: Per questi esperimenti, la linea Gal4 A9 è stato usato per guidare l&#39;espressione dei transgeni nell&#39;ala. Fly azioni possono essere ottenute presso il centro stock a Bloomington. Azioni utilizzati in questi esperimenti includono A9-Gal4 (Bl # 8761), His2Av-GFP (Bl # 5941), Sco / cyo HsCre (Bl # 1092), UAS-ChRFP-Tub (Bl # 25773), lollibow 11. 2. Selezione e Montaggio di Pupae <ol><li> Fase pupe avviene mediante la raccolta come prepupae bianco (WPP) e tenerli a 25 ° C fino a raggiungere l&#39;età appropriata o utilizzando criteri morfologici 12. </li><li> Per osservare divisioni cellulari, selezionare pupa che hanno recentemente subito la testa eversione.Da questo momento fino a poco prima eclosion (a&gt; 96 ore), pupe vengono immobile permettendo di osservazione prolungato time-lapse. </li><li> Rimuovere pupe dai flaconi per primo toccandoli con un pennello inumidito con acqua, in attesa di un minuto per permettere all&#39;acqua di allentare l&#39;adesivo e poi delicatamente sollecitazione sul pennello. </li><li> Lavare delicatamente pupe scelto da loro incitando con un pennello in acqua per eliminare i di adesivi e prodotti alimentari ghiandole salivari particelle. </li><li> Trasferimento pupe pulito per un piatto 25 millimetri Petri con un fondo coprioggetto (numero 1 ½ lamelle). </li><li> Utilizzando sottili strisce di plastilina sia o cera dentale come supporto, montare pupe modo che il tessuto di interesse è più vicino al coprioggetto. Nota: Negli studi qui descritti, sono stati osservati ali pupa, gambe, addominali o histoblasts Notum dorsale. In pratica, qualsiasi tessuto entro 20 micron della superficie del bozzolo è osservabile. </li><li> Pupe Orient attenzione in modo che il Tissue di interesse è parallela alla superficie coprioggetto di vetro. </li><li> Una volta pupe sono montati, utilizzare un pennello per trasferire un sottile strato di thiodiethylene glicole (TDG) allo spazio tra la pupa e il coprioggetto. </li></ol> Nota: TDG riduce la dispersione delle superfici, corrisponde l&#39;indice di rifrazione di olio, e permette all&#39;ossigeno di permeare il tessuto 13. L&#39;uso di oli non è consigliato, in quanto tendono a privare i tessuti di ossigeno, causando la rapida cessazione dei comportamenti cellulari. 3. Imaging Nota: Per l&#39;imaging, un microscopio confocale è probabile che sia essenziale come parafocalità rimuove la maggior parte dello sfondo oscuramento causato da intensa illuminazione del bozzolo. <ol><li> Regolare le impostazioni sulla confocale per minimizzare l&#39;impatto dell&#39;illuminazione sullo sviluppo pupa e la redditività. Per fare questo, trovare un equilibrio tra l&#39;intensità dell&#39;eccitazione e della sensibilità della raccolta. Una configurazione tipica per l&#39;imaging with Leica SP5 utilizzato lo scanner risonante (8,000 Hz), potenza del laser impostato a 2% (10% di trasmissione di potenza 20%), insieme pinhole a 120 micron, e la linea medie impostato a 8. Impostazioni variano a seconda delle condizioni sperimentali. </li></ol> Nota: Per risolvere scala multa offre 40X e 63X lenti ad immersione ad olio (0,1 millimetri distanza di lavoro) sono stati anche utilizzati con successo in questo metodo, anche se limitano la profondità di fuoco. 4. Analisi Per analizzare i frame, z-stack o film importare i file di dati FIJI, che dispone di strumenti efficaci per la visualizzazione, la misurazione e la modifica di file per la presentazione 14. Ad esempio, il tempo di completare la mitosi è stata misurata utilizzando l&#39;intervallo di campionamento e browser di immagine multidimensionale per scorrere fotogrammi durante la visione di una cellula da prophase a telofase. x, y, z-dimensioni può essere misurata utilizzando lo strumento di misura. Per le istruzioni dettagliate sul software e il suo utilizzovedi: http://fiji.sc/Fiji.

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Gli autori non hanno nulla da rivelare.

Per visualizzare, misurare e quantificare le caratteristiche delle cellule in divisione, lo sviluppo necessario di una semplice preparazione per osservare mitosi nell&#39;ala pupa vivente di Drosophila mediante analisi confocale di His2AvGFP cellule che esprimono. Questo metodo è stato usato per documentare che il ciclo cellulare nell&#39;ala pupa somiglianze forti alla cella cicli in epiteli pseudostratificato dal fatto che nuclei mossa alla superficie apicale dell&#39;epitelio cui entrano mitosi. Dopo telofa...

<html> La mosca dell&#39;aceto, Drosophila melanogaster, è un modello affermato per studiare molti aspetti della biologia. Ricerca Drosophila ha una storia ricca di sperimentazione genetica che permette forme sofisticate di manipolazione genetica, tra cui l&#39;espressione, l&#39;abbattimento e la mutazione. Con l&#39;avvento delle etichette proteine ​​fluorescenti, questo repertorio si è ampliato per includere studi sulle cellule e proteine ​​in animali vivi. L&#39;embrione mosca è un ottimo sistema per tali studi è piccolo e otticamente trasparente permettendo profondo, immagini ad alta risoluzione in vivo 1-3. Altri stadi di sviluppo mosca hanno dimostrato di essere meno trattabili, che richiedono l&#39;anestesia 4, la dissezione e la cultura a breve termine 5,6, o la creazione di finestre nella cuticola per l&#39;imaging 7,8. Queste manipolazioni solito compromettono sviluppo animale a lungo termine o influenzano l&#39;animale in modo che limitano imaging per brevi periodi. ent &quot;&gt; Per studiare nuove mutazioni in geni che assomigliano regolatori del ciclo cellulare, era essenziale trovare un&#39;adeguata preparazione per studiare la sincronizzazione e la fedeltà del ciclo cellulare. Poiché la maggior parte cicli cellulari embrionali sono troncati (SM o S-G2-M) e mutanti in studio non mostrano difetti fino fasi successive, era importante osservare il ciclo cellulare nei tessuti stadio di pupa. delle cellule epiteliali della pupa hanno un più tipico ciclo cellulare G1-S-G2-M e pupe di questa fase sono non capace di movimenti muscolari 9. Il punto di partenza iniziale per manipolazioni inclusa intatta tutta pupe esprimere Histone2AV-GFP. Nonostante l&#39;apparente opacità del bozzolo, questa preparazione intatta rivelata eccellente per lungo termine imaging in vivo. Questa tecnica è semplice basta che i ricercatori universitari abitualmente usano per studiare gli aspetti di biologia cellulare e dello sviluppo in Drosophila, eppure la risoluzione è abbastanza fine per consentire la discriminazione delle caratteristiche scala micrometrica. Con questo metodo, le osservazioni degli eventi più ore, minuti o secondi sono possibili semplicemente regolando i parametri di serie temporali. Video che utilizzano proteine, verde, giallo, arancio, rosso e blu fluorescenti, o combinazioni di questi, sono stati fatti. È importante sottolineare che, se si fa attenzione a ridurre al minimo l&#39;intensità del laser, anche di imaging a lungo termine non ha alcun effetto sullo sviluppo o la vitalità degli animali.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly stuff fly pad</td>
			<td style="width:71px;">Genesee scientific</td>
			<td style="width:119px;">59-114</td>
			<td style="width:167px;">for fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CO2 gas</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;">CD50</td>
			<td>For fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regulator</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;"> </td>
			<td>CO2 regulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly vials</td>
			<td style="width:71px;">Genesee scientific</td>
			<td>32-113RLBF</td>
			<td>Fly culture</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Drosophila lines:A9-Gal4 (Bl#8761), His2Av-GFP (Bl#5941), Sco/CyO HsCre (Bl#1092), UAS-ChRFP-Tub (Bl#25773)</td>
			<td style="width:71px;">Bloomington Stock center</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass bottom dishes #1 1/2</td>
			<td style="width:71px;">WillCo Wells BV</td>
			<td style="width:119px;"> </td>
			<td>For microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thiodiethylene Glycol</td>
			<td style="width:71px;">Fluka</td>
			<td align="right" style="width:119px;">88559</td>
			<td>mountant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Modeling clay</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>Support to position pupae against</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paintbrushes</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>To manipulate flies</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fine Forceps, Inox #5</td>
			<td style="width:71px;">Fine science tools</td>
			<td style="width:119px;">11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">computer</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>8 Gb RAM for image/movie analysis</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Fiji software</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;">Free ware http://fiji.sc/Fiji</td>
			<td>Image analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td>Any fast scanning confocal should be sufficient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20x dry, and 40x or 63x oil immersion lenses</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For imaging tissue, cellular and subcellular features</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil (non fluorescent)</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For fly manipulation</td>
		</tr>
	</tbody>
</table>

imaging through the pupal case of drosophila melanogaster

L&#39;uso di lunga data di Drosophila come modello per la biologia cellulare e dello sviluppo ha prodotto una serie di strumenti. Insieme, queste tecniche hanno permesso l&#39;analisi di biologia cellulare e dello sviluppo da diverse angolazioni metodologiche. L&#39;imaging dal vivo è un metodo emergente per osservare processi cellulari dinamici, come la divisione cellulare e la motilità cellulare. Avendo mutazioni isolato in non caratterizzate proteine ​​del ciclo cellulare putative divenne essenziale osservare mitosi in situ utilizzando l&#39;imaging dal vivo. La maggior parte degli studi di imaging dal vivo in Drosophila sono concentrati sulle fasi embrionali che sono accessibili alla manipolazione e l&#39;osservazione a causa delle loro piccole dimensioni e chiarezza ottica. Tuttavia, in queste fasi del ciclo cellulare è insolito in quanto manca di una o entrambe le fasi gap. Per contro, le cellule dell&#39;ala pupa di Drosophila hanno un ciclo tipica cellula e sottoposti ad un periodo di rapida mitosi attraversa circa 20 ore di sviluppo pupa. È facile identify e isolare pupe della fase di catturare mitosi in situ. Montaggio pupe intatta disponibile la migliore combinazione di trattabilità e durata durante l&#39;imaging, consentendo di eseguire esperimenti per diverse ore con un impatto minimo sulla vitalità cellulare e animale. Il metodo permette l&#39;osservazione di caratteristiche più piccolo, o minore di, volare cromosomi. Regolazione delle impostazioni del microscopio e dei dettagli del montaggio, ha consentito l&#39;estensione della preparazione per la visualizzazione dinamica della membrana delle cellule adiacenti e proteine ​​fluorescente come la tubulina. Questo metodo funziona per tutte le proteine ​​fluorescenti testate e in grado di catturare le caratteristiche di scala inferiori al micron su una varietà di scale temporali. Mentre limitato al esterno 20 micron della pupa con un microscopio confocale convenzionale, questo approccio all&#39;osservazione proteine ​​e dinamiche cellulari nei tessuti pupa in vivo può essere generalmente utile nello studio della biologia cellulare e dello sviluppo in questi tessuti.

Cellule in epiteli pseudostratificato, come la sviluppo occhio Drosophila, o lo strato ventricolare del sistema nervoso centrale dei vertebrati sviluppo, subiscono movimenti nucleari, definito migrazione nucleare interkinetic, in fase con il ciclo cellulare. La replicazione del DNA avviene quando i nuclei sono o vicino alla superficie basale e cellule entrano mitosi quando i nuclei raggiungono la superficie apicale 15,16. Le cellule ala pupa formano un monostrato in rapida divisione epitelio durante ...

Watch this Scientific Journal Video about Imaging Through the Pupal Case of Drosophila melanogaster at JoVE.com

Imaging Through the Pupal Case of Drosophila melanogaster

Questo documento dimostra l&#39;uso di una scansione rapida microscopio confocale a comportamenti cella di immagine direttamente attraverso il puparium. Lasciando il bozzolo intatto, questo metodo permette l&#39;osservazione e la misurazione dei processi cellulari dinamiche in una fase di sviluppo Drosophila che è difficile da studiare direttamente.

Imaging attraverso il caso pupa di<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 ...

imaging-through-the-pupal-case-of-drosophila-melanogaster

Research

JoVE Journal

Biology

Imaging Through the Pupal Case of Drosophila melanogaster

13.2K Views.  University of Miami.  Questo documento dimostra l'uso di una scansione rapida microscopio confocale a comportamenti cella di immagine direttamente attraverso il puparium. Lasciando il bozzolo intatto, questo metodo permette l'osservazione e la misurazione dei processi cellulari dinamiche in una fase di sviluppo Drosophila che è difficile da studiare direttamente. 

Video: Imaging Through the Pupal Case of Drosophila melanogaster

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.

Dissezione del sistema nervoso centrale larvali 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 ...

Ricerca

Biologia

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.

Immagini dal vivo di Drosophila melanogaster Migrazioni Hemocyte embrionali

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

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imaging verticale di Drosophila Embrioni

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

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.

Dissezione di Oenocytes da adulti 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 ...

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.

Isolamento di Drosophila melanogaster Testicoli

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

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila pupa Addome Immunoistochimica

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

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.

Misura della durata della vita 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 ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

Ex vivo Cultura della Drosophila pupa testicolo e singolo maschio cisti germinale: Dissezione, Imaging, e trattamento farmacologico

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissezione e Montaggio di<em&gt; Drosophila</em&gt; pupa Dischi Eye

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

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.

Mantenimento di un<em&gt; Drosophila melanogaster</em&gt; Cage Popolazione

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