我们非常感谢支持的BHR通过发现格兰特以及来自自然科学和加拿大工程研究理事会（NSERC）的一个研究工具和仪器格兰特。我们也承认H.小田和布卢明顿果蝇中心提供，例如实时成像序列的遗传股股票。

制备<ol><li>收集胚胎标准的葡萄汁琼脂平板 4 。它方便地使用一个自动化的果蝇卵收集器（Flymax科学器材有限公司）。使用同步上演胚胎集合将减少必须执行，以获得足够数量的胚胎在发育阶段所需的数量dechorionations。 </li><li>准备一个dechorionation幻灯片，附加了一块双面胶带载玻片;删除从磁带的支持。 </li><li>准备切割一块组织，以适应在香港总商会以及实时成像室。在精心组织和湿用蒸馏水。 </li><li>混合卤烃油（卤烃产品公司）粘度700系列和56系列在以1:1的比例。该混合物可以存储并无限期使用。 </li><li>盖玻片（22 × 40毫米，第2号）的表面上放置几小滴卤烃油。该卷是并不重要，但应在20-40μL为了。 </li></ol>程序<ol><li>随着立体显微镜的帮助下，收集胚胎使用一对珠宝商的镊子（第5号），或一个非常优良的油漆刷的葡萄汁琼脂板。胚胎往往坚持互相钳“提示;他们很容易聚集在团块。 </li><li>随着立体显微镜的帮助下，轻轻地放下，坚持到磁带表面上的dechorionation幻灯片钳提示胚胎团块。 </li><li>再次使用的体视显微镜，轻轻地微调或中风的一侧钳胚胎为了打破开放外糯绒毛膜不破裂的内在卵黄膜（如果卵黄膜破裂，胚胎破裂）。 </li><li>一旦外层的蛋壳已破裂，挑逗胚胎绒毛膜; dechorionated胚胎往往要坚持到表面的镊子。小心避免触摸磁带表面dechorionated胚胎。 </li><li>只要一个胚胎dechorionated，迅速转移到盖玻片先前准备的卤烃油下降。触摸账面dechorionated胚胎的卤烃油滴表面钳的一角 - 这个位置被驱逐出来的钳入油胚胎。 </li><li>确认胚胎转移到滴油。这是可以做到与你工作在一个黑色的背景，并使用光纤照明光源设置在一个斜角肉眼。 </li><li>试图dechorionate另一个胚胎之前，清洁镊子从任何石油。涂油钳绒毛膜表面上有较少的购买，使得dechorionation更加困难。 </li><li> Dechorionated胚胎蓬勃的卤烃油。使用产钳或画笔，并通过立体显微镜下观察时，推胚胎油价下跌的底部，并安排在所需的位置。 </li><li>快速翻转盖玻片以及在实时成像室。确认胚胎所需方向对盖​​玻片休息。由于他们在油中的浮力，胚胎会浮动盖玻片表面。盖玻片固定用胶带实时成像室。通风孔，在该地区的磁带跨越盖玻片和实时成像室以及结束之间的差距在磁带削减室。 </li><li>一个正直的荧光显微镜或荧光体视显微镜，现在可以用于图像的胚胎。 </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>

我们描述了一种实时成像分析，我们称之为悬滴协议的准备果蝇胚胎的新方法。不幸的是，它是不可能的使用悬滴协议，如果倒置显微镜工作。在这种情况下，夹层技术（抽象以上所述），必须使用压缩的胚胎仍然令人担忧。 在细胞的形状和大小进行测量，以计算力量与形态运动相关的实验，使用一个堂堂正正的显微镜和悬滴协议是可取的，因为胚胎压缩减少。此外，减少?...

标准设备，并准备活成像的胚胎，使用悬滴协议所需的材料包括以下项目：显微镜载玻片，盖玻片（22 × 40毫米，第2号），双面胶带，珠宝商的镊子（杜蒙风格第5号），卤烃油56系列和700系列（卤烃产品公司），美工刀或单边缘刀片，卫生纸，剪刀，蒸馏水，一般用途的磁带，吸管适合提供20-40μL。实时成像协议还需要一个定制的实时成像室。这是准备削减5毫米认为一个标准的显微镜幻灯片（75 x 25毫米）的尺寸聚碳酸酯塑料板和使用转子创建幻灯片（20 x 55毫米）3mm的深度抑郁。立体显微镜（解剖显微镜），还需要一个光纤照明光源。

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

绿荧光蛋白质（GFP）为基础的timelapse生活成像的动态进程，如组织形态发生，细胞与细胞间的粘附，或细胞死亡的基因调控研究的一个强大的技术。果蝇胚胎表达GFP很容易使用以立体或共聚焦显微镜成像。任何带电成像协议的目标是尽量减少不利影响，如脱水和缺氧。前准备活成像分析果蝇胚胎的协议涉及dechorionated胚胎放置在卤烃油和夹层之间的卤烃气体渗透膜和盖玻片<sup&gt; 1-3</sup&gt;。通过引进安装胚胎的压缩，以这种方式为基础的生物力学分析任何形态发生不良并发症。我们的方法，我们称之为悬滴协议，在实时成像优秀的胚胎活力的结果，不需要胚胎被压缩。简单地说，悬滴协议涉及的胚胎在下降的卤烃油是从盖玻片，这是在潮湿室位置固定，暂停的位置。除了提供气体交换，防止脱水，这样的安排利用胚胎在卤烃油的浮力，以防止他们漂流timelapse收购期间的立场。本视频介绍了如何收集和准备现场使用悬滴协议成像果蝇胚胎细节。该协议是适合使用立体显微镜或任何正直的复合荧光显微镜成像dechorionated胚胎。

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

一个简单，廉价，​​有效的方法是准备住成像分析果蝇胚胎。我们的协议提供湿度和气体交换，不压缩的果蝇胚胎。这种方法适用于基于GFP的使用立体显微镜或直立复式显微镜的果蝇胚胎的实时成像。

编制果蝇实时成像的胚胎使用悬滴协议

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 次观看.  University of Waterloo. 一个简单，廉价，​​有效的方法是准备住成像分析果蝇胚胎。我们的协议提供湿度和气体交换，不压缩的果蝇胚胎。这种方法适用于基于GFP的使用立体显微镜或直立复式显微镜的果蝇胚胎的实时成像。

视频: 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.

从Midgastrula舞台果蝇胚胎神经文化的制备

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

科研

生物学

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.

在体外分化的小鼠胚胎干细胞（MES）使用悬滴法

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.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

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.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

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.

电子显微镜对胚胎的制备果蝇胚胎心管

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.

实时成像果蝇胚胎血细胞迁移

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.

发展和成人的制备果蝇大脑和视网膜实时成像

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.

准备个人果蝇蛋钱伯斯实时成像

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.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

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.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

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