Siamo grati per il sostegno al BHR tramite una sovvenzione Discovery e Strumenti di ricerca e Grant strumento dalle scienze naturali e ingegneria Research Council del Canada (NSERC). Riconosciamo inoltre H. Oda e il Centro di Bloomington Drosophila magazzino per la fornitura di scorte genetiche che sono state usate nelle sequenze di immagini dal vivo esempio.

Preparazione <ol><li> Raccogliere embrioni su standard di succo di uva piastre di agar 4. E &#39;conveniente usare un sistema automatizzato Drosophila Egg Collector (flymax scientifico apparecchiature Ltd.). Utilizzando sincrono messo in scena le collezioni embrione ridurrà il numero di dechorionations che devono essere eseguite in modo da ottenere un numero adeguato di embrioni allo stadio di sviluppo desiderato. </li><li> Preparare una diapositiva dechorionation allegando un pezzo di nastro biadesivo ad un vetrino da microscopio, rimuovere il supporto dal nastro. </li><li> Preparare la camera di immagini dal vivo, tagliando un pezzo di tessuto per adattarsi nel pozzo della camera. Posizionare il tessuto nel bene e bagnato con acqua distillata. </li><li> Mix alocarburi olio (Halocarbon Products Corp.) serie 700 viscosità e serie 56 con un rapporto di 1:1. La miscela possono essere memorizzati e utilizzati a tempo indeterminato. </li><li> Luogo diverse piccole gocce di olio alocarburi sulla superficie di un coprioggetto (22 x 40 mm, n. 2). Il volume non è critica, ma dovrebbe essere nell&#39;ordine dei microlitri 20-40. </li></ol> Procedura <ol><li> Con l&#39;aiuto di uno stereomicroscopio, raccogliere gli embrioni dal succo d&#39;uva-agar piastre utilizzando una pinza da gioielliere (n. 5) o un pennello molto fine. Gli embrioni tendono ad attaccarsi l&#39;un l&#39;altro e per le pinze punte &#39;, sono facilmente riuniti in gruppi. </li><li> Con l&#39;aiuto di uno stereomicroscopio, abbassare delicatamente le macchie di embrioni che hanno aderito alle punte della pinza sulla superficie del nastro sul vetrino dechorionation. </li><li> Anche in questo caso con lo stereomicroscopio, delicatamente spingere o ictus gli embrioni con il lato della pinza in modo da rompere il corion esterno ceroso senza rottura della membrana interna vitellina (l&#39;embrione esploderà se la membrana vitellina è rotto). </li><li> Una volta che il corion esterno è stato rotto, stuzzicare l&#39;embrione dal corion, l&#39;embrione dechorionated tende ad aderire alla superficie della pinza. Fare attenzione a non toccare l&#39;embrione dechorionated alla superficie del nastro. </li><li> Non appena un embrione è dechorionated, rapidamente trasferimento alla goccia di olio precedentemente preparato alocarburi sul vetrino. Toccare la punta della pinza portando l&#39;embrione dechorionated alla superficie della goccia di olio alocarburi - questo sloggia l&#39;embrione dal forcipe nell&#39;olio. </li><li> Confermare il trasferimento di un embrione alla goccia di petrolio. Questo può essere fatto a occhio nudo, a condizione che si lavora su uno sfondo nero e utilizzare una fibra ottica fonte di illuminazione fissata a un angolo obliquo. </li><li> Prima di tentare di dechorionate un altro embrione, pulire qualsiasi tipo di olio dalla pinza. Pinza rivestiti in olio hanno meno di acquisto sulla superficie corion, dechorionation rendendo più difficile. </li><li> Dechorionated embrioni sono vivace nell&#39;olio alocarburi. Uso di pinze o un pennello e durante la visualizzazione attraverso uno stereomicroscopio, spingere l&#39;embrione alla parte inferiore della goccia di olio e sistemare nella posizione desiderata. </li><li> Rapidamente invertire il coprioggetto sopra il pozzo della camera di immagini dal vivo. Confermano che gli embrioni sono a riposo contro il vetrino con l&#39;orientamento desiderato. Grazie alla loro galleggiabilità in olio, gli embrioni ora galleggiano sulla superficie inferiore del coprioggetto. Fissare il coprioggetto alla camera di imaging dal vivo con del nastro adesivo. Ventilare la camera da taglio buchi nel nastro nella zona in cui il nastro si estende il divario tra la fine del vetrino e la fine del pozzo della camera di immagini dal vivo. </li><li> Un microscopio a fluorescenza in posizione verticale o uno stereomicroscopio a fluorescenza possono ora essere utilizzati per l&#39;immagine degli embrioni. </li></ol>

<ol>
	<li>Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L., Montague, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forces+contribute+to+cell+sheet+morphogenesis+for+dorsal+closure+in+Drosophila.">Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.</a> J. Cell Biol. 149, 471-490 (2000).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+processes+associated+with+germ+band+retraction+in.">Cellular processes associated with germ band retraction in.</a> , 248-2429 (2002).</li><li>Reed, B. H., Wilk, R., Schock, F., Lipshitz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-dependent+apposition+of+Drosophila+extraembryonic+membranes+promotes+morphogenesis+and+prevents+anoikis.">Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis.</a> Curr. Biol. 14, 372-380 (2004).</li><li>Wieschaus, E. p., Nusslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a practical approach. , 199-227 (1986).</li></ol>

Descriviamo un nuovo metodo di preparazione di embrioni di Drosophila per l&#39;analisi di immagini dal vivo, che noi chiamiamo il protocollo goccia. Purtroppo, non è possibile utilizzare il protocollo goccia se si lavora con un microscopio invertito. In questo caso la tecnica del sandwich (come descritto nel già astratto) deve essere utilizzato e la compressione degli embrioni rimane una preoccupazione. Negli esperimenti in cui vengono misurate la forma delle cellule e dimensione al fine ...

Dotazione di serie e materiali necessari per la preparazione di embrioni per la live-immagine che utilizzano il protocollo goccia comprendono le seguenti voci: vetrini da microscopio, coprioggetto (22 x 40 mm, n. 2), nastro biadesivo, gioiellerie forcipe (Dumont stile n. 5 ), alocarburi olio serie 56 e serie 700 (Halocarbon Products Corp.), un taglierino o singola lama di rasoio bordo, carta velina, forbici, acqua distillata, nastro scopo generale, e una pipetta adatta per la consegna di microlitri 20-40. Il protocollo di imaging dal vivo richiede anche una misura da camera dal vivo imaging. Si prepara il taglio di 5 mm pensare foglio di plastica in policarbonato alle dimensioni di un vetrino da microscopio standard (75 x 25 mm) e l&#39;utilizzo di un rotore per creare una depressione 3 millimetri nel profondo della diapositiva (20 X 55 mm). L&#39;accesso a uno stereomicroscopio (dissezione microscopio) e una fibra ottica fonte di illuminazione è anche necessario.

the preparation of drosophila embryos for live-imaging using the hanging drop protocol

Proteina fluorescente verde (GFP)-base timelapse live-imaging è una tecnica potente per lo studio della regolazione genetica dei processi dinamici come la morfogenesi dei tessuti, l&#39;adesione cellula-cellula, o morte cellulare. Embrioni di Drosophila che esprimono GFP sono prontamente ripreso utilizzando la microscopia confocale o stereoscopica. Un obiettivo di vivere-imaging protocollo è quello di minimizzare gli effetti dannosi, come la disidratazione e ipossia. Precedenti protocolli per la preparazione di embrioni di Drosophila per il live-imaging analisi hanno coinvolto messa embrioni dechorionated in olio alocarburi e sandwich di loro tra un alocarburi gas-permeabile membrana e un coprioggetto<sup&gt; 1-3</sup&gt;. L&#39;introduzione di compressione attraverso embrioni di montaggio in questo modo rappresenta una complicazione indesiderabile per qualsiasi analisi basata biomeccanica della morfogenesi. Il nostro metodo, che noi chiamiamo il protocollo goccia, i risultati ottimi della redditività degli embrioni durante immagini dal vivo e non richiede che gli embrioni da comprimere. In breve, il protocollo goccia comporta il posizionamento di embrioni in una goccia di olio Halocarbon che è sospeso da un coprioggetti, che è, a sua volta, fissa all&#39;altezza desiderata in una camera umida. Oltre a fornire lo scambio di gas e prevenire la disidratazione, tale sistema sfrutta la galleggiabilità di embrioni in olio Halocarbon per evitare che finiscano fuori posizione durante l&#39;acquisizione Timelapse. Questo video descrive in dettaglio come raccogliere e preparare gli embrioni di Drosophila per l&#39;imaging dal vivo utilizzando il protocollo goccia. Questo protocollo è adatto per gli embrioni di imaging dechorionated utilizzando stereomicroscopia o verticale microscopio a fluorescenza composto.

Watch this Scientific Journal Video about The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol at JoVE.com

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Un metodo semplice, economico ed efficace per preparare gli embrioni di Drosophila per il live-imaging analisi è presentato. Il nostro protocollo prevede l&#39;umidità e lo scambio di gas e non comprimere l&#39;embrione di Drosophila. Questo metodo è adatto per GFP-based di immagini dal vivo di embrioni di Drosophila utilizzando uno stereomicroscopio o microscopio composto verticale.

La preparazione di Drosophila Gli embrioni per Live-Imaging Utilizzando il protocollo goccia

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

the-preparation-drosophila-embryos-for-live-imaging-using-hanging

Research

JoVE Journal

Biology

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

15.0K Views.  University of Waterloo. Un metodo semplice, economico ed efficace per preparare gli embrioni di Drosophila per il live-imaging analisi è presentato. Il nostro protocollo prevede l'umidità e lo scambio di gas e non comprimere l'embrione di Drosophila. Questo metodo è adatto per GFP-based di immagini dal vivo di embrioni di Drosophila utilizzando uno stereomicroscopio o microscopio composto verticale.

Video: The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparazione delle Culture neuronale da Midgastrula fase embrioni di Drosophila

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Ricerca

Biologia

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

La differenziazione in vitro di staminali embrionali di topo (MES) Cellule con il metodo goccia

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Vivere Imaging della migrazione cellule gliali nella Eye Disco Drosophila immaginale

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparazione di embrioni per la microscopia elettronica del Drosophila Tubo cuore embrionale

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparazione di Sviluppo e degli adulti Drosophila Brains e retina per l&#39;imaging dal vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Preparazione individuale Drosophila Egg Chambers per l&#39;imaging dal vivo

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Il pomodoro / GFP-FLP / FRT Metodo per Live Imaging del Mosaico adulti<em&gt; Drosophila</em&gt; cellule fotorecettrici

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...