作者特别感谢德博拉哈里斯和Danielle麦凯。为支持这项工作提供了生物学的凯斯西储大学艺术与科学学院部，以及霍华德休斯医学研究所授予数量为52005866生物科学本科教育的支持。

1。成像方法<ol><li>在55 ° C度为20-30分钟水浴融化甘油，果冻，直到它变成一个同质的液体。漩涡，但不摇瓶，以避免形成泡沫。 </li><li>虽然果冻的融化，适用于使用一个干净的玻片上（可选）一个Kimwipe一层薄薄的甘油。 </li><li>移液器1至1.5毫升溶化甘油果冻上使用的幻灯片的表面削减P1000的枪头，创建一个薄的第一层。小心吸取试剂，以避免气泡。 </li><li>让果冻固化5分钟一个水平的表面上，然后放置在-20 ° C 5分钟的幻灯片，并进行胚胎对齐。另外，用保鲜膜覆盖的幻灯片，并在4 ° C至5天。 </li><li>移液器染色胚胎小降了70％的甘油/ PBS或其他抗淬灭甘油为基础的安装媒体（例如Slowfade或Vectashield）果冻床上用品一侧。 </li><li>开始预调胚胎单独转让果冻表面下降。他们转移使用一个倾斜的注射针，用勺子倾斜侧壁。 </li><li>大致的地方沿2〜3列的胚胎。如果一个胚胎获取滞留针，搅拌它包含其他的胚胎来释放它的下降。仔细对准他们在这一点上，不要浪费时间。列之间留一些空间。 </li><li>将胚胎滑的果冻中等水平皮下注射针头的援助。虽然滑动，东方的头和尾巴在同一方向。使他们彼此平行沿柱。删除与一块扭曲Kimiwipe的足迹留下的PBS甘油的过剩。 注意:过量的甘油会导致胚胎内凝胶松散的包裹，导致两个果冻层的分离，使其难以切割和装入。相反，清除过多的甘油就会枯竭和扁平化的胚胎。 </li><li>使用切割黄尖遍布一层薄薄的胚胎（50-150μL，对齐多少而定）的果冻。 </li><li>将幻灯片，在-20 ° C冰箱10-20分钟，或保存在4 ° C过夜。确保果冻是完全刚性的，切割前。否则，胚胎块将很难消费。切割的最好成绩是获得翌日。 </li><li>根据解剖范围，使用刀片完全对齐的胚胎两侧穿过果冻。保持在90度角直线切割刀片。这次裁员应该是非常接近的胚胎的前部和后部的两极，以避免过量的果冻，可能会阻碍成像。 </li><li>加入100-200μL冷PBT（4 ° C，PBS +0.1％吐温20）未来的削减，小心地刮去使用抹刀的胚胎之间的果冻。 注:在PBT的洗涤剂帮助果冻取消不损害胚胎的情况下的幻灯片。 </li><li>用针，转让与嵌入式胚胎进一步削减到一个干净的幻灯片的果冻块。 5-7胚胎切割更小的块。使用PBT轻松地左右移动的果冻块。另外，放置在干燥的玻璃表面块让它附着于玻璃，创造更多的精确修剪果冻，如果有必要的稳定。 </li><li>翻转直立在果冻的嵌入式刀片和针援助的胚胎。在显微镜下可视化，进行下面的两个步骤中的任一个，视显微镜上的立场正在使用。 </li><li>倒置显微镜:地方完成到盖玻片的胚胎与胚胎在直立位置，块。确保果冻量最少的胚胎方将加强与客观联系。 </li><li>立式显微镜:四个＃1盖玻片被用来作为"支持"和地方之间的"支持"，在甘油中的胚胎块用强力胶胶合两个栈。大桥的堆栈使用长盖玻片。 </li><li>共聚焦分析，检查工作要使用的目标的距离，找出是否可以形象通过胚胎或仅部分。例如，D.果蝇胚胎〜0.55毫米，长度和使用计划一个蔡司20X - APO或40X LD新福陆公司的目标可以完全成像。下面的网站有可用蔡司目标的信息，让您选择搜索目标，根据工作距离:https://www.micro-shop.zeiss.com/us/us_en/objektive.php </li></ol> 2。代表性的成果因为胚胎将保持不变，这种方法可用于精确的测量胚胎的大小和基因表达模式之间的距离。在图1中，我们显示一个核染色的DAPI（蓝色），反背蛋白（红色），SOG的mRNA（黄色）和SNA的mRNA（绿色）染色的胚盘阶段的胚胎。形态议事录SES，如内陷（图2A，B）的中胚层和中胚层细胞（图3和4）迁移也可以可视化使用此方法。不同沿DV轴Z轴位置可通过改变焦平面，如在图2-4所示。 <img src="/files/ftp_upload/2175/2175fig1.jpg" alt="图1" /> 图1。直立式光学切片一个完整的胚胎中胚阶段。mRNA和蛋白的双重染色。图中的靶mRNA 原位探测（SOG和SNA）和抗体染色的蛋白（反手背）表示。 DAPI是用来标记细胞核。注意位于顶部SOG和SNA的表达在细胞质中，而背的表达是核。 SOG新生的成绩单，位于细胞核的强有力的信号，也可以看到在这个放大倍数。 <img src="/files/ftp_upload/2175/2175fig2.jpg" alt="图2" /> 图2。一个完整的胚胎gastrulating直立成像。基因探针和各自的颜色如图所示。一）请注意，在这个阶段，三个基因相同的福陆公司（SNA，SOG和民进党，绿色），染色，表示在空间上分离的的域中。内陷中胚层表示SNA，成神经细胞层表示IND和外胚层表示， 民进党SOG的表达，而在腹侧神经系统的地区仍然存在。 B）在一个更深的共聚焦平面相同的胚胎，我们注意到强烈表示 SNA内陷中胚层的基础，但它的顶端区域表达水平较低。 <img src="/files/ftp_upload/2175/2175fig3.jpg" alt="图3" /> 图3。一个完整的胚胎与生殖带直立成像扩展 SOG RNA（黄色）; 蜗牛，IND和民进党的mRNA（绿色），背蛋白（红色）。细胞核用DAPI染色（蓝色）。一）焦平面胚胎头部附近。 B）另一个在同一胚胎的躯干地区的焦平面。注意群众中胚层细胞的横向迁移前，躺在生殖波段扩展胚胎两侧靠近腹侧中线（比较图4）。分层神经母细胞被标记为绿色（IND和SNA）。 <img src="/files/ftp_upload/2175/2175fig4.jpg" alt="图4" /> 图4。一个完整的胚胎生殖波段扩展期间 SOG RNA（黄色）; 直立成像。 蜗牛，IND和民进党的mRNA（绿色），背蛋白（红色）。 A）注分层神经母细胞从上皮细胞（箭头），染色IND和SNA都在这个阶段（绿色）。还要注意的SOG限制表达的腹侧中线（黄色）。胚胎（星号）的侧面可以看出，民进党在这个阶段中的表达。二）在同一胚胎的光学部分显示在一个胚胎的长度，对应于中后区附近。腹侧中线细胞染色SOG。

<ol>
	<li>Ay, A., Fakhouri, W. D., Chiu, C., Arnosti, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+and+analysis+for+quantifying+gene+expression+from+early+Drosophila+embryos.">Image processing and analysis for quantifying gene expression from early Drosophila embryos.</a> Tissue Engineering. 14, 1517-1526 (2008).</li><li>Gr&#946;hans, J., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+link+between+morphogenesis+and+cell+division+during+formation+of+the+ventral+furrow+in+Drosophila.">A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila.</a> Cell. 101, 523-531 (2000).</li><li>Kosman, D., Mizutani, C. M., Lemons, D., Cox, W. G., McGinnis, W., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846-846 (2004).</li><li>Witzberger, M. M., Fitzpatrick, J. A. J., Crowley, J. C., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-on+imaging:+a+new+perspective+on+dorsoventral+development+in+Drosophila+embryos.">End-on imaging: a new perspective on dorsoventral development in Drosophila embryos.</a> Developmental Dynamics. 237, 3252-3259 (2008).</li><li>Zander, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+mounting+delicate+bryophytes+in+glycerol.">On mounting delicate bryophytes in glycerol.</a> The Bryologist. 100, 380-382 (1997).</li><li>Liberman, L. M., Reeves, G. T., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+the+Dorsal+nuclear+gradient+reveals+limitations+to+threshold-dependent+patterning+in+Drosophila.">Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila.</a> Proc Natl Acad Sci U S A. 106 (52), 22317-2222 (2009).</li></ol>

以果蝇胚胎的横截面图像可以是具有挑战性，因为它需要一个 2片或切片处理，这通常是有害的荧光染色法的嵌入式媒体使用小心手清扫。另外，在共聚焦显微镜的Z -堆栈可用于截面图像三维重建。然而，这是费时，成为问题的一个精确的三维重建和漂白几个薄的共焦片。另一个值得关注的时候，成像纵向安装的胚胎是达到更深的胚胎，这也复杂荧光信号的精确测量，量化蛋白或 mRNA表达水平1的目的时，会发生光散射。即使与双光子共聚焦显微镜，光的散射仍然是一个关注，由于卵黄物质的胚胎，这使得选择最佳样品的透明度是有限的安装媒体中的不透明度。 为了规避这些问题，新技术，所谓的"最终"成像定位胚胎的垂直是最近开发的图像直播和固定样本4。在本文中，我们开发了一个垂直安装的胚胎的新协议，允许一个简单的编制和固定的胚胎组织 或染色3原位探测与多个任意大小和形状的可视化。我们的方法包括使用凝胶状的一致性用来包住对齐胚胎内安装媒体。本安装媒体包含嵌入式胚胎切割块可以放置在任何类型的共聚焦显微镜成像前直立。 嵌入到果冻的胚胎和它们垂直定位，我们可以采取横向的横截面，使用共聚焦显微镜，在沿背腹轴的胚胎细胞同时呈现。这是一个量化的目的，因为光散射和漂白的荧光染料，在传统的Z - Stack纵向安装的胚胎重建发生，现在淘汰的问题显着改善。因此，沿背腹轴的基因表达或蛋白质水平的定量准确，因为在同一时间和相同的共焦的Z -位置位于对面的背腹位置的细胞成像。此外，所描述的方法可以让我们获得一个完整的2 - D图像，而无需使用复杂的计算展开方法，可视化细胞在不同的岗位沿DV轴6的跨越，从一个3 - D胚胎背腹轴。 一些关键步骤包括调整胚胎后，删除多余的甘油，以避免分裂的果冻两层之间。然而，如果样品是太脱水，生成的图像将被畸形，并有不正确的的形态。此外，如果胚胎周围的胶状介质是减少而不是直接的角度，产生的图像可能会出现更全面，更ovular的形状，由于胚胎在90度以外的角度的位置不正确。最后，如​​果果冻是不会削减不够紧密的前/后两端的胚胎，这是不可能获得正确的图像，因为客观的工作距离，将主要用于集中到果冻，而不是胚胎。 另一个重要的一步成像后期gastrulating胚胎是认真梳理的范围下对准他们感兴趣的阶段时，可能是必要的。通常情况下，胚胎都在上演，根据形态特征，最容易在纵向位置时确认。 可以用这种方法制成的替代修改纳入防褪色剂（如苯二胺或PPD）果冻的第二层，以帮助防止荧光染料漂白。

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		<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>Glycerin Jelly mounting media</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences Company</td>
<td>17998-10</td>
<td>Contains low concentration of phenol as preservative and should be used in hood or in well ventilated area. Recipe for making your own media is described in Zander, 1997.</td>
</tr>
<tr>
<td>Zeiss LSM 700 Confocal</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>The following objectives were used: Plan-Apo 20x 0.8 M27 (WD 0.55mm); EC Plan-Neofluar 40x 1.3 oil (WD 0.21mm); LD Plan-Neofluar 0.6 Korr 40X (WD 2.9mm).</td>
</tr>
<tr>
<td>Phosphate buffered saline with 0.1% Tween 20 (PBT)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass microscope slides</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-544-7</td>
<td>&nbsp;</td>
<tr>
<td>Glass cover slips</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72204-02</td>
<td>Size 1 &frac12; (specific for the objectives used in this work).</td>
</tr>
<tr>
<td>Glass cover slips (for making supports)</td>
<td>&nbsp;</td>
<td>Fisherfinest</td>
<td>12-544-10</td>
<td>Use four coverslips glued with superglue. </td>
</tr>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>M5 Wild Heerbrugg Wild Makroskop</td>
<td>M420</td>
<td>&nbsp;</td>
<tr>
<td>Razor blade</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Steel back single edge industrial blades</td>
</tr>
<tr>
<td>Spatula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>21-401-10</td>
<td>&nbsp;</td>
<tr>
<td>Hypodermic needles</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482610</td>
<td>26 Gauge</td>
</tr>
<tr>
<td>Pipettes</td>
<td>&nbsp;</td>
<td>Labnet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

upright imaging of drosophila embryos

几家知名形成梯度和细胞运动发生沿背/腹轴<em&gt;果蝇</em&gt;胚胎。然而，使用目前的技术，以查看这些进程的比较有限。以下协议描述了一种用于安装固定和标记的新技术<em&gt;果蝇</em&gt;冠状观看共焦成像的胚胎。这种方法包括嵌入两层甘油果冻安装媒体之间的胚胎，直立定位和成像果冻条。夹在胚胎的第一步是使甘油果冻的幻灯片上的薄被褥。下一步，胚胎仔细对齐这种表面上覆盖着第二层的果冻。第二层是凝固后，果冻条被切断，翻转直立成像。介绍了替代品，用于可视化取决于要使用显微镜支架类型的胚胎。由于沿背腹轴的所有细胞都在一个单一的共焦Z平面成像的，我们的方法允许未经漂白或光散射共同纵向安装的胚胎的三维重建的荧光信号的精确测量和比较。

Watch this Scientific Journal Video about Upright Imaging of Drosophila Embryos at JoVE.com

Upright Imaging of Drosophila Embryos

在这里，我们提出了一个彩绘的安装协议<em&gt;果蝇</em&gt;在直立位置，允许使用共聚焦显微镜的横截面成像的胚胎。

直立成像果蝇胚胎

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

Research

JoVE Journal

Biology

Upright Imaging of Drosophila Embryos

9.3K 次观看.  Case Western Reserve University. 在这里，我们提出了一个彩绘的安装协议果蝇在直立位置，允许使用共聚焦显微镜的横截面成像的胚胎。

视频: Upright Imaging of Drosophila Embryos

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

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.

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 ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

收获和准备果蝇胚胎的电生理记录和其他程序

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

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 ...

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.

果蝇胚胎的体内离心

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

Primary Cell Cultures from Drosophila Gastrula Embryos

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

Primary Cell Cultures from Drosophila Gastrula Embryos

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

原代细胞培养从果蝇原肠胚

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

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...