该协议已经通过我们的工作与保罗马丁和安东尼圣哈辛托实验室内和合作。我们感谢布卢明顿联合中心，以其优良的服务和继续分享飞线果蝇社会。 BS目前由BBSRC项目赠款资助。第一次世界大战是由威康信托职业发展奖学金。

制备<ol><li>包含一个血细胞的特定Gal4的驱动程序（ 如SRP - Gal4 的 2）和无人机控制（ 如UAS - GFP）基因编码的下荧光记者获得适当的果蝇线。苍蝇SRP - GAL4 UAS - GMA 3或crq - GAL4 UAS - GFP 4，5纯合子是特别有用的成像目的（NB GMA的是绿色荧光蛋白融合moesin肌动蛋白结合结构域），参见下面的讨论Gal4的司机和UAS的范围内构造（布卢明顿联合中心包含多种）。 </li><li>通常遗传杂交进行这样突变的等位基因与Gal4的驱动程序和替代同源染色体进行的UAS构造，使用荧光平衡器 CTG 或 TTG 6平衡。这使得它可以选择无CTG / TTG相关的绿色荧光（这是在现阶段2.11）的基础上的纯合突变体。 </li><li>在与苹果汁琼脂平板7铺设笼放大的股票和地方苍蝇。苍蝇至少需要两天，使其恢复到之前铺设笼有足够多的胚胎开始被辞退。 20每个性别的苍蝇一般应足以产生足够的成像胚胎的，但应该指出的是，不同的线路有不同程度的生育。我们使用55毫米培养皿适合到塑料烧杯底部，在其基础刺破，让空气流通。胚胎收集的确切手段是不重要的，但时序是至关重要的，以便正确地收集，上演胚胎。 </li><li>收集胚胎从一个通宵的苹果汁琼脂板保持在25 ° C或从计时板。对于后者，我们通常让苍蝇打下一个预热板删除板，并放置15-16小时，在18 ° C，在安装胚胎前，前4小时，这提供了通过胚胎后期12发展阶段15。过夜板包含了更加多样化的阶段，但提供了更高水平的血细胞的荧光记者表达由于在较长的时间在25期的优势GAL4 - UAS系统° C的温度敏感。 </li></ol>程序<ol><li>打跑胚胎从苹果汁琼脂板使用了少量的水和软尖的画笔。用肉眼很容易脱落的胚胎可以看出。 </li><li>胚胎转移到一个细胞过滤器（费雪）或自制浇到，在一个烧杯中举行收集废水篮子从苹果汁琼脂平板的水篮7。 </li><li>重复步骤2.2，直到你满意为止你有足够的胚胎从苹果汁琼脂平板上转移。 </li><li>洗净胚胎细胞过滤器/水篮。 </li><li>广场细胞过滤器/苹果汁琼脂平板培养皿盖的篮子，并补充足够的整齐漂白暂停胚胎细胞过滤器/篮。 </li><li>按照下明解剖镜下的胚胎dechorionation：dechorionation完成时背的附属物已解散，这应该发生在两分钟之内。 </li><li>从漂白细胞过滤器/篮含有胚胎取出，洗掉残留的漂白剂使用水。继续到步骤2.8前应清除所有漂白剂的痕迹。一招，以评估是否所有的漂白剂已被删除印迹关闭蓝色实验室组织的残余水 - 如果有残留的漂白剂漂白蓝色白色/粉红色。 </li><li>吸干关闭剩余的水用实验室组织/细胞过滤器/篮底部的mediwipes。 </li><li>广场的水液滴在培养皿盖子。用细的画笔，从胚胎篮收集的所有的dechorionated胚胎和重新悬浮液滴。下一步胚胎干吸水使用微量或认真吸收实验室组织/ mediwipes。 </li><li>一旦胚胎已经干涸，添加voltalef油下降到涵盖所有的胚胎。将相邻液滴含有胚胎的石油第二小的下降。注：我们一直无法找到一个voltalef石油的总部设在英国的供应商;卤烃油700（Sigma公司）可以用来代替。 </li><li>在一个荧光解剖镜下选择适当上演胚胎所需的油滴的制表大师钳的一双（5号）的基因型。这些镊子应以舀起他们的卵黄膜穿刺胚胎弯曲向内（图1）。选定的胚胎转移到第二油滴。重要的是，你能够看到荧光血细胞，在解剖显微镜，以便能收集到的良好形象，共聚焦显微镜（图2）。我们通常安装阶段13/14胚胎形象腹侧中线或15阶段的胚胎血细胞的横向迁移以下散布在胚胎的血细胞的活力形象。 </li><li>坚持一个Petriperm / Lumox的菜（Sarstedt）底面的两个盖玻片（18x18mm，厚度为1），使用2 voltalef石油小滴，留下约之间（图3）1厘米;，这些将被用来支持放置在盖玻片胚胎，所以不粉碎他们。 Petriperm菜（直径50mm），包含一个疏水性，透气膜。我们发现的菜肴变得更容易使用，一旦被多次使用（菜，可消灭70％的乙醇和重用）。 </li><li>在解剖镜下明，拿起选定胚胎弯曲钳和行腹侧逐一平行于盖玻片边缘（图3）。这是可能的对齐，以这种方式在15胚胎，这取决于你的灵巧和耐心。重要的是轻轻地操纵胚胎，胚胎和Petriperm盘膜是脆弱的，可以很容易破裂。 </li><li>一旦胚胎是一致的添加油小滴，让它蔓延，形成了两者之间的盖玻片的同质层。油后已蔓延（这可能需要几分钟）检查胚胎腹侧起来。如果胚胎已经稍微卷起，再次重新定位他们与镊子。 </li><li>最后，使用镊子（3号）通过胚胎盖玻片（18x18mm，厚度为1），休息后，先前坚持的两个盖玻片。胶盖玻片盖玻片支持使用指甲油（图3）。 </li><li>安装胚胎共聚焦或宽视场显微镜Petriperm盘和安装在使用适当的适配器阶段Petriperm菜。直立或倒置显微镜可用于通过盖玻片（而不是通过膜）与客观对焦镜头。 </li></ol>代表性的结果： 本协议描述了如何安装果蝇胚胎血细胞的胚胎的腹侧实时成像。如果做得正确，这将是容易产生血细胞或者剧照或电影。的主要因素是用于图像的血细胞（尤其是物镜）显微镜，但获得的图像的性质也将取决于所处的发展阶段，温度胚胎提出了在使用和GAL4和UAS线。 荧光蛋白的表达水平较高，使血细胞成像更容易，因此重要的是能够看到的血细胞在胚胎阶段的协议（图2包含的例子清晰的血细胞在胚胎内，采取2.11相机装上一个解剖镜下）。 GAL4和UAS的构造，因此人数的增加，使一个更大的信号信噪比。此外，这减少了需要高的激光强度或增加曝光时间的成像，这反过来又使血细胞的行为时，时间较长。 GFP的表达非常高的水平，将显示血细胞形态的细节，特别是薄片状片状，环绕圆形胞体（图4A - B）。不包括GFP圆形区域代表吞噬体（图4A - C的）。片状（图4B）中出现的丝状伪足，手指也可以看出。两个Gal4的司机仍然足以看到这些进程（图4C），尤其是当一个或多个SRP - Gal4的 （见讨论），但扫描速度较慢或更大的激光功率的激光共聚焦显微镜可能是必需的。随着表达水平的降低，它变得更加难以形象的血细胞的突起;尽管如此，它仍然是可以跟踪这些条件下的血细胞迁移，细胞体内仍然明显，即使突起不太清楚（图4D）。 在发展的早期阶段（高达阶段13）血细胞迁移到另一个密切联系，它往往是很难区分单个细胞。 13血细胞的阶段结束，已经形成了一个下来的腹侧中线（图5A）的单行，然后，变得更加运动型，迁移横向腹神经索（图5B）的边缘。血细胞内的动态突起的肌动蛋白细胞骨架，可以直接观察到通过GMA（图5C）或樱桃moesin表达。 安装在这样的胚胎，使气体交换，防止脱水和胚胎以下成像仍然可行。如果胚胎是在安装时一般是明显的，因为胚胎的内容将通过其卵黄膜泄漏的损坏。如果一个胚胎开始脱水，则往往被视为在卵黄膜的变形。偶尔胚胎期间将推出一个timelapse电影的过程中，然而，这往往是更长的时间尺度电影的问题。 LastlY，一次安装的胚胎，使实验者的完美的方向在他们的实验中获得胚胎的最好机会。 <img src="/files/ftp_upload/1696/1696fig1.jpg" alt="图1" /> 图1。钳dechorionated胚胎操纵 。 制表大师钳（大小5）提示应弯曲向内，以时尚的一个工具舀如下所示的胚胎。外表面的弯曲区域也是有用的操纵胚胎Petriperm膜定位时，因为他们拥有没有锋利的边缘，可以穿刺胚胎。 <img src="/files/ftp_upload/1696/1696fig2.jpg" alt="图2" /> 图2。代表图像的胚胎，将产生良好的生活成像效果。 voltalef石油dechorionated胚胎（2.11协议的阶段）上的荧光解剖显微镜拍摄的图像。 crq GAL4，UAS - GFP的胚胎 ;阶段13（A）和阶段15（二）SRP - GAL4 UAS - GFP横向意见。 （三）在其中的一个阶段15 SRP - GAL4 UAS - GFP /侧视图; crq Gal4的，uas-GFP/uas-N17Rac胚胎血细胞有没有迁移的头，这表明什么时血细胞的胚胎像沿着其迁徙路线并不明显。侧视图一个阶段17 SRP - GAL4，UAS - GFP; crq - GAL4 UAS - GFP发展（d）在这个阶段的胚胎肠道令人费解的结构，肌肉收缩的发病防止超出了这个胚胎的实时成像发展阶段。腹阶段，13（E）和14期的意见（F） 的SRP - GAL4 UAS红毒刺胚胎散布血细胞，用荧光标记的核。观察荧光血细胞在2.11的协议阶段是一个先决条件，以获得优异的图像;前是为所有图像的权利。 <img src="/files/ftp_upload/1696/1696fig3.jpg" alt="图3" /> 图3。胚胎上安装一个Petriperm / Lumox的菜 。 18x18mm盖玻片（厚度1）坚持底面Petriperm使用的油的小水滴，约1cm分隔的菜。胚胎，然后列队腹侧长（前后轴）平行的盖玻片的边缘，并覆盖着石油的一个小水滴。一旦石油已蔓延至填补第三的盖玻片（18x18mm厚度1）使用以前坚持的两个盖玻片作为一个桥梁，以防止被压扁了的胚胎油覆盖的胚胎上轻轻放在两个盖玻片之间的差距。这盖玻片，然后粘到两个盖玻片桥梁使用两个小滴指甲油。设置后，胚胎可以直立或倒置显微镜盖玻片（而不是通过Petriperm膜），重点是通过与客观镜头成像。 <img src="/files/ftp_upload/1696/1696fig4.jpg" alt="图4" /> 图4。代表从绿色荧光蛋白标记的血细胞实时成像结果。 Z型血细胞上的一个阶段14 SRP - GAL4 UAS - GFP的腹侧方; crq GAL4 UAS - GFP胚胎（AB），预测。 （一）是一个较低的放大倍率的形象，如用于监测timelapse电影中的血细胞的发育迁移。 （B）是一个更高的放大倍率腹侧中线血细胞，显示其形态精致的细节。 （C）是一个1米的血细胞，在一个阶段SRP - GAL4 UAS - GFP / 14的腹侧中线片; crq - Gal4的，UAS - GFP / +胚胎，揭示Gal4的司机和UAS的拷贝数较低结构也足以产生的良好形象。 （四）显示在一个阶段14 crq GAL4 UAS - GFP胚胎的血细胞ž投影。血细胞突起不太明显，由于GFP表达较低，但它仍然是可能使电影和跟踪这个Gal4的驱动程序和UAS的组合构建的血细胞迁移。徕卡LSM510共聚焦显微镜图像;前是在所有图像;引起卵黄膜的自体荧光图像的边缘的环。 <img src="/files/ftp_upload/1696/1696fig5.jpg" alt="图5" /> 图5。代表的GMA表达血细胞实时成像结果 。 阶段13 ​​腹侧中线（A）和14期的血细胞的Z -预测（二）SRP - GAL4，UAS - GMA的胚胎，取自timelapse电影，显示血细胞的发育迁移。肌动蛋白动力学的详细信息，可以得到更高的GMA表达血细胞（三）放大成像。 GMA组成的GFP融合moesin和标签微丝肌动蛋白结合域。前路是在所有的图像，图像共聚焦显微镜。

<ol>
	<li>Brand, A. H., 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=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PDGF/VEGF+receptor+controls+blood+cell+survival+in+Drosophila.">The PDGF/VEGF receptor controls blood cell survival in Drosophila.</a> Dev Cell. 7, 73-84 (2004).</li><li>Dutta, D., Bloor, J. W., Ruiz-Gomez, M., VijayRaghavan, K., Kiehart, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+morphogenetic+movements+in+Drosophila+using+Gal4-UAS-driven+expression+of+GFP+fused+to+the+actin-binding+domain+of+moesin.">Real-time imaging of morphogenetic movements in Drosophila using Gal4-UAS-driven expression of GFP fused to the actin-binding domain of moesin.</a> Genesis. 34, 146-151 (2002).</li><li>Stramer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+wound+inflammation+in+Drosophila+embryos+reveals+key+roles+for+small+GTPases+during+in+vivo+cell+migration.">Live imaging of wound inflammation in Drosophila embryos reveals key roles for small GTPases during in vivo cell migration.</a> J Cell Biol. 168, 567-573 (2005).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+embryonic+haemocytes:+masters+of+multitasking.">Drosophila melanogaster embryonic haemocytes: masters of multitasking.</a> Nat Rev Mol Cell Biol. 8, 542-551 (2007).</li><li>Halfon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+fluorescent+protein+reporters+for+use+with+the+Drosophila+Gal4+expression+system+and+for+vital+detection+of+balancer+chromosomes.">New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.</a> Genesis. 34, 135-138 (2002).</li><li>Sullivan, W., Ashburner, M., Hawley, R. S. <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 protocols. , (2000).</li><li>Tepass, U., Fessler, L. I., Aziz, A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+hemocytes+and+their+relationship+to+cell+death+in+Drosophila.">Embryonic origin of hemocytes and their relationship to cell death in Drosophila.</a> Development. 120, 1829-1837 (1994).</li><li>Millard, T. H., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+filopodial+interactions+during+the+zippering+phase+of+Drosophila+dorsal+closure.">Dynamic analysis of filopodial interactions during the zippering phase of Drosophila dorsal closure.</a> Development. 135, 621-626 (2008).</li><li>Doerflinger, H., Benton, R., Shulman, J. M., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+PAR-1+in+regulating+the+polarised+microtubule+cytoskeleton+in+the+Drosophila+follicular+epithelium.">The role of PAR-1 in regulating the polarised microtubule cytoskeleton in the Drosophila follicular epithelium.</a> Development. 130, 3965-3975 (2003).</li><li>Olofsson, B., Page, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Condensation+of+the+central+nervous+system+in+embryonic+Drosophila+is+inhibited+by+blocking+hemocyte+migration+or+neural+activity.">Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity.</a> Dev Biol. 279, 233-243 (2005).</li><li>Paladi, M., Tepass, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+Rho+GTPases+in+embryonic+blood+cell+migration+in+Drosophila.">Function of Rho GTPases in embryonic blood cell migration in Drosophila.</a> J Cell Sci. 117, 6313-6326 (2004).</li><li>Vlisidou, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+embryos+as+model+systems+for+monitoring+bacterial+infection+in+real+time.">Drosophila embryos as model systems for monitoring bacterial infection in real time.</a> PLoS Pathog. 5, e1000518-e1000518 (2009).</li><li>Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin-based+epithelial+adhesion+and+cell+matching+during+Drosophila+dorsal+closure.">Dynamic actin-based epithelial adhesion and cell matching during Drosophila dorsal closure.</a> Curr Biol. 10, 1420-1426 (2000).</li><li>Wood, W., Jacinto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+movement+during+dorsal+closure+in+Drosophila+embryos.">Imaging cell movement during dorsal closure in Drosophila embryos.</a> Methods Mol Biol. 294, 203-210 (2005).</li><li>Kunwar, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tre1+GPCR+initiates+germ+cell+transepithelial+migration+by+regulating+Drosophila+melanogaster+E-cadherin.">Tre1 GPCR initiates germ cell transepithelial migration by regulating Drosophila melanogaster E-cadherin.</a> J Cell Biol. 183, 157-168 (2008).</li></ol>

此过程中最重要的因素是健康的胚胎有明确标记的血细胞的选择和安装不小心损坏。一旦胚胎中的卤烃油，脱水和抗一旦安装了好几个小时可以成像。在我们的手中，我们可以形象的血细胞，三个小时，胚胎或明显的照片损害可以忽略不计的脱水，采取每三分钟我们蔡司LSM510共聚焦显微镜图像与40X目标的Z - Stack。由于血细胞是高度动态的，可能有一个空间和时间分辨率之间的权衡：如果需要一个?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>70&mu;m pores</td>
</tr>
<tr>
<td>Halcarbon oil 700</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H8898</td>
<td>&nbsp;</td>
<tr>
<td>Lumox/Petriperm dish</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>96077305</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

live imaging of drosophila melanogaster embryonic hemocyte migrations

许多研究解决细胞迁移<em&gt;在体外</em&gt;方法，是生物体本身，而有关生理环境。在这里，我们提出了一个协议的安装<em&gt;果蝇</em&gt;胚胎及随后的荧光标记血细胞实时成像，这个有机体的胚胎巨噬细胞。利用GAL4 - UAS系统<sup&gt; 1</sup&gt;我们的驱动器的多种荧光标记基因编码，在血细胞的标记，按照他们在整个胚胎发育疏散的表达。以下收集所需的发展阶段的胚胎，外层的绒毛膜被删除，胚胎，然后安装在卤烃油之间的疏水性，透气膜和实时成像的玻璃盖玻片。此外，如速度和方向性更高分辨率的成像，再加上使用的F -肌动蛋白和微管可以提供更详细的信息，关于这些细胞骨架的动态的荧光记者毛候鸟参数。

Watch this Scientific Journal Video about Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations at JoVE.com

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

果蝇血细胞分散在整个胚胎发育。此协议演示如何安装和图像这些用荧光标记的血细胞的胚胎的迁移。

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 ...

live-imaging-of-drosophila-melanogaster-embryonic-hemocyte-migrations

Research

JoVE Journal

Biology

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

15.6K 次观看.  University of Bath. 果蝇血细胞分散在整个胚胎发育。此协议演示如何安装和图像这些用荧光标记的血细胞的胚胎的迁移。

视频: Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

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 ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

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 GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

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  ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...