我们感谢施洛米Krispin神经管​​形象。特别是，我们承认尤瓦肉桂启动GFP转染技术。这项工作得到了来自以色列科学基金会，协会法国反对LES肌病，德意志Forschungsgemeinshaft，合作研究中心488的赠款为CK

1。准备的微量移液器我们Narishige PP - 83标准的吸管拉马拉我们的吸液管与热定型为13.5。精确的设置必须校准，目的是在枪头直径8至10微米。我们使用长丝含有硼硅玻璃管，外径1.0毫米，内径0.75-0.78毫米。移液器因此存储在一个塑料菜与菜的边缘接触保护提示。 2。 DNA的制备利益的质粒转染到大肠杆菌DH5α中使用标准程序的大肠杆菌。从单菌落过夜培养用于纯化质粒最大制备试剂盒（应该做的任何商业套件）。我们使用质粒浓度为1.0微克/μL非克隆注射。高浓度的增加枪头堵塞。对于克隆的应用，质粒浓度要低得多，必须进行调整（见下文）。对于pCAGG - GFP，生产克隆成果的最佳浓度被发现约0.05-0.1微克/μL。 1 - 2μL的质粒通常用于每个实验，在实验开始前，添加少量的快速绿色粉末（坚持干尖，锋利的玻璃吸管）质粒。质粒是在台式离心机最大速度为2分钟离心。这个过程似乎减少吸管堵塞问题。 3。注射胚胎 3.1胚胎的制备我们使用注射鹌鹑，以及鸡蛋。卵孵化躺在其长轴上，直到所需的发育阶段。鸡蛋是用70％乙醇擦洗，并让其干燥。鸡蛋指出到底是刺穿手术剪（这是必要的，只有在鸡蛋）和1毫升（鹌鹑）或2毫升蛋白（鸡）是一个10毫升的注射器（19G针）从鸡蛋绘制。该洞，然后用热石蜡密封。 实际注射前，打开一个窗口，在鸡蛋壳，用手术剪顶部。配售的超额壳胶带切割，并通过它可以帮助减少从壳碎片。含有抗生素的PBS缓冲液，吸管立即进入卵子。要访问的胚胎，卵黄和/或羊水膜切割和挪用使用0.14毫米的昆虫装到一个持针器引脚。无毒的墨水（鹈鹕＃17）是注入帮助可视化胚胎的胚盘底，用火，拉毛细管装在一个抽吸。 3.2加载和安装的微量拉马微量可装载与质粒拉火毛细血管足够的直径，装到一个抽吸。少量的质粒绘制到开幕相反的吸管，以削尖的提示使用毛细管和加载，并尽可能接近由内而外的一角。因此移液器装上手动的空气压力，喷油器上安装一个显微。施加最小的压力，如果必要的话，为了让质粒的解决方案，以达到吸管尖。 3.3质粒注射吸管是操纵鸡蛋的液体介质，同时运用一些空气压力，以防止吸头堵塞。有关组织域刺穿。压力变化的提示进入组织往往是足够的释放与可见的快速绿色质粒。如果是这样的情况下，适用于轻微的压力注射器，直到弹出质粒/快绿。这个过程可以反复沿轴的胚胎，例如在相邻的体节，或沿神经管的程度。注射之后，PBS可能会增加。窗口是用胶带密封（封口膜是不可取的，特别是鹌鹑蛋，它会增加胚胎死亡率，可能阻止通过外壳的氧气交换）。返回到处理的胚胎孵化器。避免通风，并放置在孵化器中的水的小容器，因为它们有助于保持水分和增加产量。 4。调整注入参数和监测成功率 Asessing和校准各种注塑参数是瞄准单细胞分析应用的关键。每次注射的事件会导致一个单细胞的成功标签。质粒浓度过高会导致非克隆的标签，而高失败率非常低的浓度结果。克隆注射注入质粒的浓度必须优化。我们建议整个纯化质粒股票的稀释，而不是预先确定的浓度稀释个人PREPS。后者可能会引入一些成功率，可能是由于在纯度上的差别，以及其他参数，如超螺旋质粒的比例差异。 4.1评估整装观测的成功率整体式观察，可以执行注射胚胎的初步评估，根据荧光的双眼。注射后6至8小时充足的评估与pCAGG增强型绿色荧光蛋白构建成功率。这是提供目标网站是肤浅的不够是显而易见的（如体节的背，背神经管，外胚层等，见图1A - C）。这一步是特别有助于评价实验系列参展要么太多标记的细胞，或者没有任何荧光细胞。如果需要进一步孵化，含抗生素的PBS液中应加入鸡蛋后查看密封胶条应更换。 4.2串行节的分析评估克隆评估注射成功率的最佳手段是通过连续切片注入胚胎，采用免疫组织化学可视化的标记细胞。在这些条件下，孵化时间短，如注射后4小时。胚胎是固定的，石蜡切片（或cryosectioning）处理。玻璃安装部分，然后去paraffinized记者可视化免疫染色。如生物素 - 亲和扩增方法可能是可取的。最后，连续切片扫描检测注入段标记的细胞。如果经初步试验，而不是单个细胞的多个获得，必须稀释质粒股票和反复的过程，直到达到足够的结果。成功率过高的情况下，可能需要2至10倍的质粒原液稀释。 对于每个质粒转染，彻底的校准，以确保克隆应该被重复。 5。代表性的成果我们通常开展pCAGG - GFP每体节的单次注射，同时针对每胚胎1 2 6-12段。此设置下，一个明确的信号，可以检测到整个装载观察8小时注射后，至少在一个段的十一个胚胎。在这些条件下，标签，进一步证实被克隆（图1B，C）。最胚胎使用DNA浓度高时，可能会出现至少有一个成功的注入，提高了多个单元格已转的可能性。 部分序列分析表明，克隆转染成功率达到10％左右。这意味着，以实现克隆，技术是每个定义，效率非常低。另一方面，在这些条件下，超过93％的正面标签事件被发现被克隆1。 <img src="/files/ftp_upload/2133/2133fig1.jpg" alt="图1" /> 图1。克隆到神经和骨骼祖细胞注射的pCAGGS - GFP（AC）显示背神经管（新台币）的标签（一），dermomyotome（DM）（二）腹外域，或中央的DM表（三）横截面，pCAGGS - GFP的显微注射到一个祖后6小时。 （四）中央DM产生在两个真皮（四），以及肌肉（M）（见文献1的细节）的衍生物，单祖细胞克隆转染后的两天。

<ol>
	<li>Ben-Yair, R., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+analysis+of+the+avian+dermomyotome+sheet+reveals+the+existence+of+single+cells+with+both+dermal+and+muscle+progenitor+fates.">Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates.</a> Development. 132, 689-701 (2005).</li><li>Ben-Yair, R., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+and+bone+morphogenetic+protein+differentially+act+on+dermomyotome+cells+to+generate+endothelium,+smooth,+and+striated+muscle.">Notch and bone morphogenetic protein differentially act on dermomyotome cells to generate endothelium, smooth, and striated muscle.</a> J Cell Biol. 180, 607-618 (2008).</li><li>Cinnamon, Y., Kahane, N., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+early+development+of+specific+hypaxial+muscles+from+the+ventrolateral+myotome.+.">Characterization of the early development of specific hypaxial muscles from the ventrolateral myotome. .</a> Development. 126, 4305-4315 (1999).</li><li>Stark, D. A., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+green+fluorescent+protein+as+a+single-cell+marker+in+living+embryos.">Photoactivatable green fluorescent protein as a single-cell marker in living embryos.</a> Dev Dyn. 233, 983-992 (2005).</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70, 148-154 (2002).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Hewapathirane, D. S., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation+in+vivo+within+the+intact+developing+brain.">Single cell electroporation in vivo within the intact developing brain.</a> J Vis Exp. , (2008).</li><li>Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat Biotechnol. 20, 87-90 (2002).</li><li>Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+integration+and+conditional+expression+of+electroporated+transgenes+in+chicken+embryos.">Stable integration and conditional expression of electroporated transgenes in chicken embryos.</a> Dev Biol. 305, 616-624 (2007).</li></ol>

上面介绍的方法是修改为先前所描述的技术，提供亲脂性染料的小细胞亚群或3个单祖。它有三个以上的标准电技术的主要优势。首先，它提供了一个标签在靶组织中的信心离散网站的手段，让远远大于电空间分辨率（例如图1B，C）。其次，它允许单一细胞的标签，从而使跟踪的血统，另有 1 2（图1C，D） ​​的正常条件下在体内。三，基因表达怀念在单细胞水平宗族隔离，...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Pipette tubes</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>B100-75-10</td>
<td>&nbsp;</td>
<tr>
<td>Capillaries</td>
<td>&nbsp;</td>
<td>Drummond Scientific</td>
<td>2-000-100</td>
<td>&nbsp;</td>
<tr>
<td>Insect needles</td>
<td>&nbsp;</td>
<td>Watkins &amp; Doncaster</td>
<td>E6871</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep solution</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>03-031-1B</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

single cell transfection in chick embryos

发育生物学的一个中心主题是多样化的谱系和澄清的分子机制。这需要一个正常和实验条件下的单细胞的命运的透彻分析。为此，目标单祖细胞的转染方法，是一个先决条件。我们在这里介绍一个简单的方法转染在OVO鸡组织中单个细胞，让可靠的谱系跟踪以及遗传操作技术。体节或神经管内，是针对特定的组织域的DNA直接注入利益的上皮细胞，导致零星的单细胞转染。记者，无性系种群可能因此被追踪长达三天，并与对照相比，无性系种群的基因操纵行为可以。此方法利用鸡胚无障碍的优势，以及与新兴的基因操纵的工具。我们比较，并讨论其优点被广泛使用的电击方法，以及可能的应用，还建议使用额外的体内模型。我们提倡使用这种方法的重要补充和补充现有的谱系追踪和基因干扰工具。

Watch this Scientific Journal Video about Single Cell Transfection in Chick Embryos at JoVE.com

Single Cell Transfection in Chick Embryos

使用精尖微量注入鸡体节或神经管的子域的质粒DNA。质粒的浓度调整，以产生单一的转染细胞。然后，我们允许的细胞发育成无性系种群。

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

single-cell-transfection-in-chick-embryos

Research

JoVE Journal

Biology

11.0K 次观看.  Hadassah Medical School - Hebrew University. 使用精尖微量注入鸡体节或神经管的子域的质粒DNA。质粒的浓度调整，以产生单一的转染细胞。然后，我们允许的细胞发育成无性系种群。

视频: Single Cell Transfection in Chick Embryos

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

科研

生物学

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.

在OVO的HH阶段10个鸡胚Electroporations

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 ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

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 ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.