这项研究是由一个国家卫生研究院（NIH）的核心格兰特（5P30EY008098）及以下的资金XW：美国国立卫生研究院批准R01EY016099和研究，以防盲职业发展奖的支持。我们感谢协助共焦成像基拉Lathrop。

第1天。制备 1。设置鱼杂交在晚上设置成对杂交斑马鱼。为了防止随机交配前所需的时间，使用分频器分开，在交配磁带的雌性和雄性的鱼。绿色荧光蛋白表达的转基因鱼，用于捐助卵裂球的来源。 pt104转基因株系（邹等 ，2008），用在这里，表达GFP的无所不在EF1启动子的控制下。 GFP的表达，使供体细胞的可视化实时成像。其他表达合适的荧光蛋白的转基因鱼线，可用于特定需求。 2。准备胚胎控股楔形琼脂糖井培养皿中，倒入融化，准备在E3蛋水（韦斯特，2007年），1％琼脂糖。飘起了一个塑料模具，琼脂糖溶液楔形突起，让琼脂糖溶液完全凝固。小心取出模具和E3的水浸泡琼脂糖床。 3。准备移植针使用在电源设置11.5针拉玻璃毛细管移液器（世界精密仪器）一个垂直进针拉马（大卫KOPF仪器）。良好的针尖应与长轴的锥度。在一个45度角的针尖研磨与一个微型磨床（Marishige）（图1A），直到40 -45μm，内径斜面开口。清理针尖，针的钝端连接一根软管，软管连接到一个注射器，然后大吃，并释放了10％的氢氟酸溶液，直到所有可见的碎片被删除。接下来，用蒸馏水洗净的技巧，其次是100％的乙醇。空气干燥，直到所有的乙醇蒸发的针。重要的是要确保提示完全干燥：残留的乙醇会毁在高温抛光步骤针。软化和波兰的针尖粗糙的边缘，一个微型的伪造者的灯丝（Marishige）所产生的热量的烘烤针​​尖。不要让针尖在抛光过程中接触的灯丝。虽然长丝及长丝和针尖之间的距离传递电力的数量可以调整，以达到所需加热的条件下。温度不宜过高：这会熔化和变形的针尖。后粗糙的边缘平滑，仔细触摸针尖加热灯丝，然后轻轻拉动针灯丝产生尖端（图1B）。一个尖锐，光滑，清洁的针是获得完整的捐助者的卵裂球，不损害主机胚胎的关键。 4。准备一个胚胎定位探头拉过来一个玻璃吸管气体火焰获得精细玻璃探头与理顺结束。这个探测器将用于定位在正确的方向前移植的胚胎。 第2天。卵裂球移植 1。收集和dechorionate胚胎第二天，在跨磁带删除的分隔，让鱼交配。放置在培养皿中的胚胎充满了E3的水。然后，通过手动撕裂，一双细镊子从胚胎的chorions dechorionate胚胎。使用弯曲轴的玻璃吸管，仔细转移到琼脂糖井dechorionated胚胎。避免任何与空气中的胚胎接触，导致爆裂的胚胎。让胚胎发育为28.5 °琼脂糖水井中的C，直到受精后3小时，或HPF。 2。成立了移植仪器虽然胚胎的发展，成立了由矿物油填充的微泵系统移植针连接到一个移植仪器。确保系统不漏，无气泡被困在系统。回填土与矿物油整个移植针，除了为0.5 - 1μL的空间，在尖端张开。 3。移植卵裂球移植卵裂球，用一个玻璃定位探头方向的3 - 4 - HPF胚胎的卵裂球的一侧向上。然后，轻轻插入一个捐助者的胚胎移植针，慢慢地吸进针5-20卵裂球。插入主机胚胎的卵裂球填充针，并慢慢释放的卵裂球。会影响他们的后代在后来的发育阶段的本地化的卵裂球被释放的网站。为了分析视网膜和大脑发育，释放细胞在动物极。在移植过程中，确保针尖不接触空气。为了最大限度地减少机械损伤，主机胚胎应留在28.5 ° C，直到第二天在琼脂糖井。然后，Transfer镶嵌胚胎到24孔板中，用0.5毫升1％琼脂糖预涂，每口井在一个胚胎。 E3蛋水1毫升，每孔加入。为了防止细菌感染，加10μL100X青霉素和链霉素E3蛋水。 第3天。马赛克胚胎的嵌入和实时成像 1。嵌入胚胎准备在E3蛋水1.5％低熔点琼脂糖（LMP），并保持在30℃水浴的解决方案。的马赛克胚胎转移到一个在0.17毫米玻璃底部厚度FluoroDish组织培养皿（世界精密仪器）。除去多余的E3水离开刚才的E3少量所涉及的胚胎。 30-50μL融化了1.5％LMP胚胎。快速定位的眼睛或大脑尽可能接近的玻璃底前琼脂糖溶液凝固。溶液冷却下来后，添加额外的琼脂糖溶液，以进一步巩固在胚胎。然后，浸泡2-3毫升E3蛋水的凝固的琼脂糖块。 2。共焦成像共聚焦显微镜，一个水浸泡倒置的目标与大聚焦深度（奥林巴斯，PlanApo，40 X/0.90 WLSM，日本）使用。一滴水添加到目标，然后放置一个FluoroDish组织培养皿上的目标。图像的发展，执行的胚胎，每5分钟拍照时间推移成像。根据实验的性质，镜头之间的时间间隔应进行调整。 代表性的成果图2显示了有代表性的24 HPF镶嵌斑马鱼。 GFP表达突出绿色的供体细胞。视网膜神经上皮细胞覆盖整个视网膜壁的厚度。 影片中展示了24 HPF主机大脑的供体细胞的运动。公告细胞形态随时间变化的。 <img src="/files/ftp_upload/1924/1924fig1.jpg" alt="图1" /> 图1移植针尖的形状。答：一针针尖斜面开放氢氟酸清洗后的碎片。 B.尖端产生的针尖。 <img src="/files/ftp_upload/1924/1924fig2.jpg" alt="图2" /> 图2。可视化绿色荧光蛋白表达的供体细胞嵌合斑马鱼的胚胎在24 HPF。左侧面板显示绿色荧光蛋白在大脑和视网膜的信号。面板中间是一个明亮的胚胎领域的图片。右侧的面板显示合并后的图像。 电影1。现场GFP阳性的供体细胞成像显示出广泛的在发展过程中大脑的神经上皮细胞的形态变化。电影中的个别帧，共收集了24和​​25 HPF之间，每<a href="http://www.jove.com/files/ftp_upload/1924/1924_Wei_Movie1.wmv">5分钟，点击</a>这里看电影。

<ol>
	<li>Zou, J., Lathrop, K., Sun, M., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+RPE+maintained+by+Nok+is+essential+for+retinal+epithelial+polarity+and+cellular+patterning+in+zebrafish.">Intact RPE maintained by Nok is essential for retinal epithelial polarity and cellular patterning in zebrafish.</a> Journal of Neuroscience. 28 (50), 13684-13695 (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> THE ZEBRAFISH BOOK. , (2007).</li><li>Ho, R. K., Kane, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+action+of+zebrafish+spt-1+mutation+in+specific+mesodermal+precursors.">Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors.</a> Nature. 348, 728-730 (1990).</li><li>Catalano, A., Raymond, P., Goldman, D., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dou+yan+Mutation+Causes+Patterning+Defects+and+Extensive+Cell+Death+in+the+Retina.">Zebrafish dou yan Mutation Causes Patterning Defects and Extensive Cell Death in the Retina.</a> Developmental Dynamics. 236 (5), 1295-1236 (2007).</li></ol>

在这段视频中，我们展示了一个卵裂球移植的方法来分析在斑马鱼的视网膜和大脑发育。此协议也可以用来分析其他组织和器官的发展，在主机的胚胎捐赠者卵裂球，只需释放到相应的位置。与绿色荧光蛋白分子的标签产量比用荧光染料标记的葡聚糖（何和凯恩1990年）标签的背景清洁。这是因为右旋糖酐染料从死亡的供体细胞的碎片持续更长的时间比绿色荧光蛋白，从而推断成像（卡塔拉...

E3的蛋水： <ul><li> 5毫米氯化钠</li><li> 0.17 mM的氯化钾</li><li> 0.33毫米氯化钙2 </li><li> 0.33毫米硫酸镁4 </li><li> 0.1％亚甲蓝</li></ul>

transplantation of gfp-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos

在自己的位置和财产广泛胚胎发育过程中细胞的变化。这些变化是受细胞和它们的环境之间的相互作用。嵌合胚胎的遗传背景不同的细胞组成的，是伟大的工具来研究基因介导的细胞 - 细胞相互作用。斑马鱼胚胎早期发育阶段的透明度，允许在细胞水平上的组织和器官的形态发生的直接可视。在这里，我们展示了一个协议，以产生在斑马鱼胚胎的嵌合体视网膜和大脑的供体细胞进行实时成像。该协议包括移植针，准备移植绿色荧光蛋白GFP阴性主机，表达捐助卵裂球和捐助现场共聚焦显微镜下的细胞行为的审查。稍作修改，这个协议也可以被用来研究的其他组织和机关在斑马鱼的胚胎发育。使用GFP标签的供体细胞的优点进行了讨论。

Watch this Scientific Journal Video about Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos at JoVE.com

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

我们展示了协议，实时成像胚胎发育过程中的细胞行为产生的嵌合斑马鱼的胚胎。

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

transplantation-gfp-expressing-blastomeres-for-live-imaging-retinal

Research

JoVE Journal

Biology

10.6K 次观看.  University of Pittsburgh. 我们展示了协议，实时成像胚胎发育过程中的细胞行为产生的嵌合斑马鱼的胚胎。

视频: Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

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 ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

斑马鱼化工屏幕自动成像和分析的发展。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

鳉胚胎和细胞移植嵌合体生成Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

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  ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.