Questo lavoro è stato sostenuto da NIH concedere NS27501 a DKOD. Supporto aggiuntivo per questo lavoro da un HHMI professor Grant a DKOD.

I. Drosophila di raccolta degli embrioni <ol><li> Preparare piatti raccolta delle uova <ol><li> Fate bollire 200 ml di dH 2 O con 8 g di agar </li><li> Raffreddare a 50 ° C </li><li> Aggiungere 2 ml di EtOH e 2 ml di acido acetico </li><li> Versare in 35 piatti di plastica millimetri Petri, top </li><li> Raffreddare e conservare in un contenitore a tenuta d&#39;aria a 4 ° C </li><li> Fa circa 80 tavole </li></ol></li><li> Preparare pasta lievito <ol><li> Sciogliere il lievito attivo Fleishmans cottura in acqua per pasta </li><li> Microonde per 20-30 secondi per uccidere i lieviti (attenzione ai bollente) </li><li> Conservare in siringa da 10 cc (senza ago) a 4 ° C per 2 settimane </li></ol></li><li> Mosche: Gli adulti utilizzati per la raccolta delle uova sono generalmente più produttivi tra i 3 ei 14 giorni dopo emergenti, a condizione che essi hanno avuto accesso a cibi freschi. Molti stock mutante hanno ridotto la fertilità che può manifestarsi in un tasso ridotto di deposizione delle uova e un cambiamento l&#39;età in cui sono più produttivi. In generale, diverse centinaia di adulti fanno per una buona deposizione delle uova della popolazione. </li><li> Procedimento: Stendere uno strato sottile di lievito pasta su una piastra di raccolta delle uova. Ciò fornisce un substrato favorevole per la posa delle uova. Adulti trasferimento vola una bottiglia pulita raccolta delle uova e nastro il piatto delle offerte alla bocca della bottiglia. Non lasciare le mosche in queste bottiglie più di 3-4 ore, come l&#39;umidità aumenta e le mosche si blocca in pasta e sui lati della bottiglia. </li></ol> II. Dechorionation embrione con candeggina <ol><li> Impostare un imbuto Buchner collegato ad un pallone filtrante collegato ad un aspiratore. Mettete un pezzo di carta Whatman n. 1 nell&#39;imbuto. Con aspirazione in funzione, bagnare la carta con acqua distillata sterile. </li><li> Sciacquare embrioni dalle piastre di raccolta sulla carta da filtro con un flusso di acqua da una bottiglia di lavaggio. </li><li> Sciacquare in diversi volumi di acqua sterile, spegnere aspiratore, e quindi aggiungere un volume imbuto del 50% di candeggina. Lasciate stare le uova 5-10 minuti. Il chorions sarà sciolto in 1-2 minuti, ma il più a lungo si lasciano gli embrioni in candeggina più del lievito contaminante verranno distrutti. Scegliere qualsiasi adulti o delle larve che hanno sciacquato piatto. </li><li> Sciacquare gli embrioni in una piastra di Petri sterile (NON coltura tissutale) con acqua distillata sterile. Molti degli embrioni che si attacchi al fondo del piatto se non è stato pre-bagnata. Sciacquare più volte con acqua sterile in questo piatto. </li><li> Esaminare gli embrioni a luce trasmessa di cogliere metà gastrula embrioni palco. </li></ol> III. Preparazione delle colture singolo embrione <ol><li> Fai DDM1 (vedi le ricette nella sezione IV) </li><li> Versare l&#39;acqua al largo piatto di embrioni appena prima di coltura. Coprire gli embrioni con i media sterile. </li><li> Di cui 3, 35 mm piastre Petri. Mettere in 4 coprioggetto autoclave tondo in ogni piatto. Su ogni vetrino, mettere 5 ml di mezzo. Usa Bellco coprioggetto-bassa coverlips vetro al piombo. Bellco biologica Vetro 800-257-7043 Cat # 1943-00012 </li><li> Tirare qualche pipette abbastanza bene. Le pipette da noi utilizzati sono tirato su un PP-83 estrattore Narishige. Le dimensioni esatte della pipetta non sono cruciali e noi di solito li tira più fine che vogliamo e li rompere la punta nello stesso campo di vista come l&#39;embrione per misurare le dimensioni. Se non si desidera per aspirare tutti gli embrioni in una volta sola, e il vostro non vogliono la punta così piccolo che ci vogliono 5 minuti per aspirare le cellule. Puntare ad una via di mezzo. Esaminare gli embrioni a luce trasmessa e utilizzare un tubo di aspirazione collegato alla bocca la pipetta per rimuovere il contenuto dell &#39;embrione. Disperdere le cellule sul vetrino preparato con delicatezza espellere le cellule più vicino possibile al coprioggetto possibile. Si consiglia di esaminare i vetrini a più alto potere e disperdere ulteriormente le cellule nello stesso modo. </li><li> Lasciate che le cellule sedersi per non più di 10 minuti. Inondare il piatto con i media e messo in incubatrice, 4-5% CO 2 ambiente, se si utilizza un terreno specifico. Temperatura tra 22-24 ° C. Noi abitualmente uso 23 ° C e 95% di umidità. L&#39;umidità è importante, come si vuole evitare l&#39;evaporazione. È possibile utilizzare uno standard di incubatore di mammifero posto in un frigorifere con controllo della temperatura impostata a 23 ° C. </li></ol> IV. DDM1 un terreno specifico per la coltivazione delle culture <ol><li> Fai DMEM: A 100 ml di Ham F12/DME Media (DMEM) (Irvine Scientific # 9052). <ol><li> Aggiungere 0,476 g Hepes (20mm) (Sigma # H-3375). Mix. </li><li> Aggiungere 1,25 ml di L-Glutammina 200 mm (Irvine Scientific # 9317). Mix. </li><li> Filtro in cappa con filtro 0,2 micron siringa acetato (bisogno di 2 filtri). </li><li> Il pH è 6.64 e Osm 288. </li><li> Fai aliquote di 10 ml in provette da 15 ml. </li><li> Conservare a 4 ° C per non più di 2 settimane. Nota: utilizzare sempre in autoclave acqua filtrata e fare in contenitori che sono riservati per i Media e supplementi solo. Non mettere mai un elettrodo pH o mescolate a barre utilizzati per altri scopi in Media.To fare DDM1: integratori Aggiungi elencati di seguitodi DMEM appena prima coltura. </li></ol></li><li> A 10 ml di DMEM aggiungere: <ul><li> 100 ul transferrina </li><li> Putrescine 100 microlitri </li><li> Selenio 100 microlitri </li><li> Progesterone 100 microlitri </li><li> 50 microlitri insulina </li></ul></li></ol>

<ol>
	<li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic-AMP-dependent+plasticity+at+excitatory+cholinergic+synapses+in+Drosophila+neurons:+Alterations+in+the+memory+mutant+dunce.">Cyclic-AMP-dependent plasticity at excitatory cholinergic synapses in Drosophila neurons: Alterations in the memory mutant dunce.</a> J. Neurosci. 20, 2104-2111 (2000).</li><li>Hodges, D. D., Lee, D., Boswell, K., Preston, C. F., Hall, L. M., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tipE+regulates+sodium-dependent+repetitive+firing+in+Drosophila+neurons.">tipE regulates sodium-dependent repetitive firing in Drosophila neurons.</a> Mol. Cell Neurosci. 19, 402-416 (2002).</li><li>Lee, D., Su, H., Dowd, O., K, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+receptors+containing+Rdl+subunits+mediate+fast+inhibitory+synaptic+transmission+in+Drosophila+neurons.">GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons.</a> J. Neurosci. 23, 4625-4634 (2003).</li></ol>

Nelle culture Drosophila preparati da embrioni fase midgastrula i neuroni derivano da precursori neuroblasti, molte delle quali dividono prima differenziazione in vitro. Questo sistema offre così un&#39;occasione unica per esplorare i fattori genetici e ambientali importanti in fasi molto precoci dello sviluppo neuronale (Rohrbaugh et al., 2003). Abbiamo dimostrato che i neuroni coltivati ​​in un mezzo semplice definito differenziarsi in neuroni elettricamente eccitabili, che formano anche funzionale connessioni sinaptiche (O Dowd, 199...

Le culture fatte in questo mezzo deve essere mantenuta in un 5% di CO2 incubatore a circa 22-24 ° C. Noi usiamo una cultura standard di tessuti di mammiferi incubatore posto in una stanza fredda e riscaldata a 23 ° C. Sempre usato in autoclave acqua filtrata e fare in contenitori che sono riservati per i media e gli integratori solo. Non mettere mai un pHmetro o mescolate a barre utilizzati per altri scopi nei media.

preparation of neuronal cultures from midgastrula stage drosophila embryos

Questo video illustra la procedura per le colture primarie di neuroni da midgastrula fase embrioni di Drosophila. I metodi per la raccolta di embrioni e la loro dechorionation con candeggina sono dimostrati. Utilizzando una pipetta di vetro collegata ad un tubo di aspirazione bocca, ci illustrano la rimozione di tutte le cellule da embrioni singolo. Il metodo per disperdere le cellule di ogni embyro in una piccola (5 l) goccia di media su un vetrino di vetro non rivestito è dimostrata. Una vista attraverso il microscopio a 1 ora dopo placcatura illustra la densità delle cellule preferito. La maggior parte delle cellule che sopravvivono quando coltivate in un terreno specifico sono neuroblasti che dividono una o più volte nella cultura prima di estendere i processi neuritiche da 12-24 ore. Una vista attraverso il microscopio illustra il livello di crescita dei neuriti e la ramificazione prevista una sana cultura a 2 giorni in vitro. Le culture sono cresciute in una semplice base di bicarbonato un terreno specifico, in un 5% di CO 2 incubatore a 22-24 ° C. Processi neuritiche continuare a elaborare oltre la prima settimana di cultura e quando entrare in contatto con neuriti da cellule vicine spesso forma funzionale connessioni sinaptiche. I neuroni in queste culture, si manifestano voltaggio-dipendenti di sodio, calcio e canali del potassio e sono elettricamente eccitabili. Questo sistema di coltura è utile per lo studio molecolare dei fattori genetici e ambientali che regolano il differenziamento neuronale, eccitabilità, e formazione di sinapsi / funzione.

Watch this Scientific Journal Video about Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos at JoVE.com

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

Questo video dimostra la preparazione di colture primarie di neuroni da midgastrula fase embrioni di Drosophila. Vedute di colture vive visualizzare le celle 1 ora dopo la placcatura e neuroni differenziati dopo 2 giorni di crescita in un medium a base di bicarbonato definite. I neuroni sono elettricamente eccitabili e formare connessioni sinaptiche.

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 ...

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8.7K Views.  University of California, Irvine (UCI). Questo video dimostra la preparazione di colture primarie di neuroni da midgastrula fase embrioni di Drosophila. Vedute di colture vive visualizzare le celle 1 ora dopo la placcatura e neuroni differenziati dopo 2 giorni di crescita in un medium a base di bicarbonato definite. I neuroni sono elettricamente eccitabili e formare connessioni sinaptiche.

Video: Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Culture primarie neuronale dal cervello di tardo fase Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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Biologia

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

Posizionamento Growth Factor-Coated Perline su embrioni di pollo Stadio

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

In Electroporations Ovo della Fase HH 10 embrioni di pollo

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Colture cellulari neuronali da Aplysia per imaging ad alta risoluzione di coni di crescita

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

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

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

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

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

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 ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparazione del Aplysia Senso-motorio colture cellulari neuronali

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

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 ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In vivo centrifugazione di embrioni di Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

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 ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Colture cellulari primarie da Drosophila Gastrula embrioni

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...