Este trabalho foi financiado pelo NIH para conceder NS27501 DKOD. Suporte adicional para este trabalho a partir de uma HHMI Professor Grant para DKOD.

I. de colheita de embriões Drosophila <ol><li> Prepare pratos de coleta de ovos <ol><li> Ferva 200 ml dH 2 O com 8 g de agar </li><li> Arrefecer a 50 ° C </li><li> Adicionar 2 ml EtOH e 2 ml de ácido acético </li><li> Despeje em 35 milímetros petri pratos de plástico, tops </li><li> Esfriar e guarde em recipiente apertado do ar a 4 ° C </li><li> Faz cerca de 80 placas </li></ol></li><li> Prepare colar Yeast <ol><li> Dissolver o fermento Fleishmans ativa fermento em água para formar colar </li><li> Microondas por 20-30 segundos para matar levedura (atente para ferver mais) </li><li> Loja em seringa de 10 cc (sem agulha) a 4 ° C por 2 semanas </li></ol></li><li> Moscas: Os adultos utilizados para a coleta de ovos são geralmente mais produtiva entre 3 e 14 dias depois de sair, desde que tenham tido acesso a alimentos frescos. Muitas ações têm mutante diminuição da fertilidade, que pode se manifestar de uma taxa reduzida de postura de ovos e uma mudança na idade em que eles são mais produtivos. Geralmente, várias centenas de adultos fazem para uma população postura boa. </li><li> Procedimento: Espalhe uma camada fina de pasta de levedura em uma placa de coleta de ovos. Isto fornece um substrato favorável para a postura dos ovos. Adulto transferência voa para uma garrafa limpa ovo recolha e tape o prato de coleta para a boca da garrafa. Não deixe as moscas nessas garrafas mais do que 3-4 horas como a umidade aumenta e as moscas ficam presas na pasta e nas laterais do frasco. </li></ol> II. Dechorionation embrião com Bleach <ol><li> Criar um funil de Buchner ligado a um frasco de filtro ligado a um aspirador. Coloque um pedaço de papel Whatman # 1 no funil. Com sucção em execução, molhe o papel com água destilada estéril. </li><li> Enxágüe embriões a partir de chapas para a coleta de papel de filtro com uma corrente de água de uma garrafa de lavagem. </li><li> Lave-os em vários volumes de água estéril, desligue aspirador, em seguida, adicionar um volume funil de água sanitária a 50%. Deixe os ovos sentar 5-10 minutos. O córions será dissolvido em 1-2 minutos, mas quanto mais tempo você deixar os embriões no bleach mais da levedura contaminante será destruída. Escolher os adultos e as larvas que têm lavado prato. </li><li> Enxágüe os embriões em uma placa de Petri estéreis (NÃO cultura de tecidos) com água destilada estéril. Muitos dos embriões grude no fundo do prato, se não tiver sido pré-molhada. Enxaguar várias vezes com água estéril neste prato. </li><li> Examine os embriões com luz transmitida para escolher embriões meados de gástrula palco. </li></ol> III. Preparação de culturas único embrião <ol><li> Faça DDM1 (ver receitas na seção IV) </li><li> Derramar água fora prato de embriões pouco antes de cultivo. Cobrir embriões com meios estéreis. </li><li> Conjunto com 3, 35 pratos de petri milímetros. Coloque em 4 autoclavado lamínulas redondas em cada prato. Em cada lamela, colocar 5 mL de meio. Use Bellco lamínulas de baixa coverlips vidro de chumbo. Bellco Biológica Copos 800-257-7043 Cat # 1943-00012 </li><li> Puxar alguns pipetas razoavelmente bem. O pipetas que usamos são puxados em um extrator Narishige PP-83. As dimensões exatas da pipeta não são cruciais e que geralmente puxá-los mais finos do que queremos e eles quebram a ponta no mesmo campo de visão como o embrião para medir o tamanho. Você não quer para sugar todos os embriões de uma só vez, e seu não quer a ponta tão pequena que leva 5 minutos para sugar as células. Apontar para algures no meio. Examine os embriões com luz transmitida e usar um tubo de sucção da boca ligado ao pipeta para remover o conteúdo do embrião. Dispersar as células para a lamínula cuidadosamente preparado por expelir as células tão perto da lamela possível. Você pode querer examinar as lamelas com maior poder e mais dispersar as células da mesma maneira. </li><li> Permitir que as células sentar-se por não mais que 10 minutos. Flood o prato com a mídia e colocar em incubadora, de 4-5% CO 2 ambiente, se estiver usando meio definido. Temperatura entre 22-24 ° C. Rotineiramente use 23 ° C e umidade de 95%. Umidade é importante, como você quiser evitar a evaporação. Você pode usar uma incubadora de padrão de mamíferos colocado em uma das câmaras frias com controle de temperatura definida para 23 ° C. </li></ol> IV. DDM1 meio definido para o cultivo das culturas <ol><li> Faça DMEM: Para 100 mls de F12/DME Ham Media (DMEM) (Irvine Scientific # 9052). <ol><li> Adicionar 0,476 g Hepes (20mM) (Sigma # H-3375). Mix. </li><li> Adicionar 1,25 ml L-Glutamina 200 mM (Irvine Scientific # 9317). Mix. </li><li> Filtro na capa com 0,2 m de acetato de seringa filtro (precisa de 2 filtros). </li><li> O pH é 6,64 e Osm 288. </li><li> Faça alíquotas de 10ml em tubos de centrífuga de 15ml. </li><li> Armazenar a 4 ° C por não mais de 2 semanas. Nota: Utilize sempre água filtrada e fazer autoclavado em recipientes que são reservados para Mídia e suplementos apenas. Nunca coloque um eletrodo de pH ou agitar bar utilizado para outros fins em Media.To fazer DDM1: suplementos Adicionar listados abaixopara DMEM pouco antes de cultivo. </li></ol></li><li> A 10 ml de DMEM acrescentar: <ul><li> 100 mL Transferrina </li><li> 100 mL Putrescine </li><li> 100 mL Selenium </li><li> 100 mL de progesterona </li><li> 50 mL de 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>

Em culturas de Drosophila preparados a partir de embriões em estágio midgastrula os neurônios surgem a partir de precursores neuroblastos, muitos dos quais divide antes de diferenciação in vitro. Este sistema, portanto, proporciona uma oportunidade única para a exploração de fatores genéticos e ambientais importantes em fases muito iniciais de desenvolvimento neuronal (Rohrbaugh et al., 2003). Nós temos mostrado que os neurônios cultivados em um meio simples definida diferenciar em neurônios eletricamente excitáveis ​​que t...

As culturas feitas neste mídia deve ser mantida em uma incubadora de 5% CO2 em cerca de 22-24 ° C. Usamos um padrão de cultura de tecidos de mamíferos incubadora colocado em uma sala fria e aquecida a 23 ° C. Sempre usada água filtrada e fazer autoclavado em recipientes que são reservados para a mídia e os suplementos apenas. Nunca coloque um medidor de pH ou agitar bar utilizado para outros fins nos meios de comunicação.

preparation of neuronal cultures from midgastrula stage drosophila embryos

Este vídeo ilustra o procedimento para a tomada de culturas primárias de neurônios a partir de embriões midgastrula estágio Drosophila. Os métodos de coleta de embriões e sua dechorionation o uso de alvejante são demonstradas. Usando uma pipeta de vidro ligado a um tubo de sucção da boca, que ilustram a remoção de todas as células de embriões individuais. O método para dispersar as células de cada embrionários em uma queda (5 l) pequenas de meio sobre uma lamela de vidro sem revestimento é demonstrada. Uma visão através do microscópio, 1 hora após o plaqueamento ilustra a densidade celular preferido. A maioria das células que sobrevivem quando cultivada em meio definido são neuroblastos que dividem uma ou mais vezes na cultura antes de estender processos neuríticas por 12-24 horas. Uma visão através do microscópio ilustra o nível de crescimento de neuritos e ramificação esperado em uma cultura saudável de dois dias in vitro. As culturas são cultivadas em um bicarbonato simples, baseada médio definido, em um 5% CO 2 incubadora a 22-24 ° C. Processos neuríticas continuar a desenvolver durante a primeira semana de cultura e quando fazem contato com neurites de células vizinhas que muitas vezes forma funcional conexões sinápticas. Neurônios nessas culturas, se manifesta de sódio voltagem dependentes, cálcio e canais de potássio e são eletricamente excitáveis. Este sistema de cultura é útil para estudar molecular fatores genéticos e ambientais que regulam a diferenciação neuronal, excitabilidade, e formação de sinapses / função.

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

Este vídeo demonstra a preparação de culturas primárias de neurônios a partir de embriões midgastrula estágio Drosophila. Pontos de vista de culturas vivas mostram as células de 1 hora após o plaqueamento e neurônios diferenciados após 2 dias de crescimento em um meio de bicarbonato baseado definido. Os neurônios são eletricamente excitáveis ​​e formam conexões sinápticas.

Preparação de culturas de neurônios a partir de embriões Midgastrula Stage 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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Biology

8.8K visualizações.  University of California, Irvine (UCI). Este vídeo demonstra a preparação de culturas primárias de neurônios a partir de embriões midgastrula estágio Drosophila. Pontos de vista de culturas vivas mostram as células de 1 hora após o plaqueamento e neurônios diferenciados após 2 dias de crescimento em um meio de bicarbonato baseado definido. Os neurônios são eletricamente excitáveis ​​e formam conexões sinápticas.

Vídeo: 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.

Culturas primárias neuronais do cérebro de Tarde Stage Drosophila pupas

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

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.

Fator de Crescimento colocando-Revestido Beads em embriões de galinha Early Stage

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.

Em Electroporations Ovo de Estágio HH 10 embriões de galinha

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.

Culturas de células neuronais de Aplysia para imagens de alta resolução de Cones de Crescimento

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 Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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.

Elaboração de Drosophila Os embriões para Live-Imaging Usando o protocolo gota suspensa

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.

Preparação de Aplysia Sensório-motor Neuronal culturas de células

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.

Preparação de embriões de Microscopia Eletrônica do Drosophila Tubo cardíaco embrionário

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 Centrifugação de Embriões 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.

Imagem vertical de Drosophila Os embriões

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