We thank Dr. Gaudenz Danuser and members of his lab for creating the plusTipTracker software and for helpful discussion regarding using the software, in particular Maria Bagonis and Sebastien Besson. We especially thank the Boston College Media Center for their assistance and support in the creation and editing of the video. We also thank members of the Lowery Lab for useful discussions and constructive criticism, and Abigail Antoine for proof-reading the manuscript. This work was funded by an NIH R00 MH095768 award to LAL.

该协议与视频是为了作为一个伴侣到原来的文件，说明更详细地4软件包，以及附带的软件下载Danuser实验室网站上的技术报告。我们鼓励读者如果有使用该软件对其他问题仔细审查这些文件。 1，在此之前的图像分析<ol><li>每个定时影片转换成TIFF（标记图像文件格式）图像文件的顺序。如果有多个生长锥/单元在给定的电影，第一茬每一个生长锥/格打造自己的图像序列。 注意：这是没有必要的，因为各个区域，其中感兴趣区（ROI），可以在plusTipTracker来选择。然而，使用较小的图像尺寸增大了运算处理的速度，所以此步骤，建议如果在图像中显著空白空间。 </li><li>保存每个系列的TIFF在自己的在MATLAB中设置访问路径中被称为“图像”文件夹（注意，“图像”是区分大小写）。要添加一个新的路径，浏览到相关的文件目录中的“当前文件夹”窗口，在该目录图标上单击右键，选择“添加到路径 - 选定的文件夹和子文件夹”。重要的是，该plusTipTracker软件文件夹被添加到路径，以及。 </li></ol> 2 plusTipGetTracks 注：在图像分析中的第一步骤是检测EB1-GFP彗星，彗星链接到轨迹，并确定微管动力学的参数。这是获得与命令“plusTipGetTracks”4。 <ol><li>要开始分析，开放式的MATLAB应用程序，并输入“plusTipGetTracks”进入命令行窗口。这将导致出现一个新的对话框。 </li><li>点击“设置新项目”，然后选择一个（或MOR先前的TIFF图像序列，通过选择适当的“图像”文件夹（或包含“图片”文件夹）目录E）。一旦这一步完成后，文件目录（roi_1）将被创建（在保存“图像”在同一个文件夹），将包含未来的数据文件。注：在“设置新项目”中的步骤可以提前完成，一个单独的会话中。 </li><li>一个新的窗口将出现：“选择一个多边形，在最后一点上单击右键，然后单击”创建规则&#39;“。点击“确定”。所选择的图像序列的第一个图像将被显示出来。使用鼠标点击，并创建一个多边形，它包括生长锥的全部内容。双击鼠标，关闭该多边形。 </li><li>一旦多边形被关闭，一个对话框将会出现：“你想选择其他的投资回报率？”如果图像有另一个生长锥来分析，选择“是”;否则Š选“否”。 </li><li>选择要立即分析该项目。点击“选择项目”，然后选择文件夹（roi_X）来分析。 </li><li> àlistSelectGUI画面会出现。选择从屏幕的左侧的项目（次）和将它们移动到右侧的画面。点击“确定”。选择一个位置保存项目列表，然后单击“保存”。 </li><li>选择“检测”，“跟踪”和“后处理”。一旦这些选择已经作出，右侧的对话框将成为可配置的。配置每个选项。 <ol><li>这些参数被用于检测的彗星链接到MT的轨道。在选择这些控制参数进行跟踪详情载于随附的软件下载技术报告的PDF 9-10页;仔细阅读本报告，如果遇到问题。跟踪EB1-GFP的彗星在Xenop的目的我们蟾生长锥，使用下面的跟踪设置：搜索半径范围（像素）5-12，最小分履带长度（帧）3;最大间隙长度（帧）8;最大收缩率因数0.8，最大倾角前50，最大角度10落后，波动半径2.5。这些设置被显示在图1。 </li></ol> 注：最大收缩率设定为减少的检测“向后差距”的数目，如“向后差距”不在的生长锥的上下文来分析是有用的，给定的拥挤状况和可能的错误在轨道之间的联系。此外，无论是最大前进角和波动半径设定为相对较高，为生长锥的MT表现除了生长和收缩率小的频繁易位，并增加这些控制设置允许在连接这一步增加运动。 <ol><li>在后处理设置填充取决于所需的特定图像采集的设置。 </li></ol> &lt;/ LI&gt; <li>一旦设置已配置，点击“开始”。取设定已选择的软件的运行。这可能需要几分钟到几小时，这取决于所选择的项目和它们的大小的数目。在命令窗口中会显示剩余的每个函数的估计时间。当plusTipGetTracks步骤完成后，在命令窗口中会显示“完成！” 注：MT动力学具体参数现在已经计算了该软件的长名单（见参考文献4的细节），但对于MT动态的生长锥的分析，最有用的参数来检查是MT增长的轨道速度（以微米/分钟），增长的轨道寿命（以秒为单位），并且增长的轨道长度（微米）。 </li></ol> 3 plusTipSeeTracks 注意：我们的微管轨道已经被定义，则函数“plustipSeeTracks”用于轨道可视化4。此功能可以提供用于可视化多个输出时，包括空间MT动力学的地图和速度的电影，但这里的重点是仅仅使用“音轨影”以显示叠加在生长锥图像MT轨道。虽然plusTipGetTracks可以同时分析多个电影，plusTipSeeTracks只能分析一次一个电影。 <ol><li>输入“plusTipSeeTracks”进入命令行窗口。 </li><li>在对话框中加载后，点击“选择项目”。选择包含​​该项目的可视化，并单击“选择文件夹”的父目录。一个新的窗口将出现“选择您希望显示的项目”。选择可视化，并单击“确定”的文件。 </li><li>接下来，点击“选择保存的投资回报率。”导航到同一个文件夹roi_X在上一步中选择一个，并选择名为“roiYX”的文件。 </li><li>点击“选择输出目录”指定瓦特这里的MATLAB将节省的轨道可视化文件。注意：我们建议您使用包含数据的其余部分相同的文件夹中。 </li><li>选择“Make轨道电影”，并在屏幕中会显示所有与+提示计算彗星轨道plusTipGetTracks。这一步可以节省的跟踪时间序列在电影格式，文件“allTracks_X_X_X”中。有用于保存电影为AVI选项，否则默认格式是作为Quicktime.mov文件。 </li></ol> 4 plusTipGroupAnalysis 注意：此最终功能是用于创建动画的群体进行分析和他们的MT轨道参数的比较。 <ol><li>输入“plusTipGroupAnalysis”进入命令行窗口。要手动选择组进行比较，首先取消选择“自动组从层次结构”。然后，点击“选择项目”。导航到包含所有roi_X文件夹的父目录来分析。 </li><li> àlistSelectGUI画面会出现。选择所有的项目中的基团，从屏幕的左侧包括与它们移动到右侧的画面。点击“确定”。选择一个位置保存项目列表，然后单击“保存”。 </li><li>将出现一个窗口：“请从列表中选择第一集团”。点击“确定”。该listSelectGUI窗口将再次显示。这一次，只选择那些符合应当汇集起来的第一批文件。点击“确定”。 </li><li>然后，输入组名称，然后单击“确定”。将出现一个窗口：“选择另一个组？”答案据此，继续选择组。将出现一个窗口：“选择一个位置来保存您的群组列表”。导航到位置，然后单击“保存”。 </li><li>点击“选择输出目录”，选择其中的输出文件夹将存储。 </li><li>选择要进行哪种类型的组的分析 - MT的磁道是否应该合并的每个组或每个细胞分析应该执行。推荐的统计测试已经指定。以包含所有的分析磁道，取消选择“删除在电影的开始/结束磁道”。否则，有此复选框选中删除所有MT增长的轨道是在过程的影片开始或结束。 </li><li>本集团的分析做出选择后，选择“比较组”。 </li></ol>

<ol>
	<li>Stepanova, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+microtubule+growth+in+cultured+neurons+via+the+use+of+EB3-GFP+(end-binding+protein+3-green+fluorescent+protein).">Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein).</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 2655-2664 (2003).</li><li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microtubule+plus+end+tracking+protein+Orbit/MAST/CLASP+acts+downstream+of+the+tyrosine+kinase+Abl+in+mediating+axon+guidance.">The microtubule plus end tracking protein Orbit/MAST/CLASP acts downstream of the tyrosine kinase Abl in mediating axon guidance.</a> Neuron. , 913-926 (2004).</li><li>Purro, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+regulates+axon+behavior+through+changes+in+microtubule+growth+directionality:+a+new+role+for+adenomatous+polyposis+coli.">Wnt regulates axon behavior through changes in microtubule growth directionality: a new role for adenomatous polyposis coli.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28, 8644-8654 (2008).</li><li>Applegate, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=plusTipTracker:+Quantitative+image+analysis+software+for+the+measurement+of+microtubule+dynamics.">plusTipTracker: Quantitative image analysis software for the measurement of microtubule dynamics.</a> Journal of structural biology. 176, 168-184 (2011).</li><li>Lowery, L. A., Faris, A. E., Stout, A., Van Vactor, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Explant+Cultures+from+Xenopus+laevis.">Neural Explant Cultures from Xenopus laevis.</a> Journal of visualized experiments : JoVE. (68), e4232 (2012).</li><li>Long, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+analysis+of+CLASP-interacting+protein+functions+during+interphase+microtubule+dynamics.">Multiparametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics.</a> Molecular and cellular biology. 33, 1528-1545 (2013).</li><li>Myers, K. A., Applegate, K. T., Danuser, G., Fischer, R. S., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+ECM+mechanosensing+pathways+regulate+microtubule+dynamics+to+control+endothelial+cell+branching+morphogenesis.">Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis.</a> The Journal of cell biology. 192, 321-334 (2011).</li><li>Nishimura, Y., Applegate, K., Davidson, M. W., Danuser, G., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+screening+of+microtubule+growth+dynamics+identifies+MARK2+as+a+regulator+of+leading+edge+microtubules+downstream+of+Rac1+in+migrating+cells.">Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.</a> PLoS One. 7, e41413 (2012).</li><li>Akhmanova, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+ends:+a+dynamic+protein+network+controls+the+fate+of+microtubule+tips.">Tracking the ends: a dynamic protein network controls the fate of microtubule tips.</a> Nature reviews. Molecular cell biology. 9, 309-322 (2008).</li><li>Schuyler, S. C., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+'plus-end-tracking+proteins':+The+end+is+just+the+beginning.">Microtubule 'plus-end-tracking proteins': The end is just the beginning.</a> Cell. 105, 421-424 (2001).</li><li>Mitchison, T., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+instability+of+microtubule+growth.">Dynamic instability of microtubule growth.</a> Nature. 312, 237-242 (1984).</li><li>Mimori-Kiyosue, Y., Shiina, N., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+behavior+of+the+APC-binding+protein+EB1+on+the+distal+ends+of+microtubules.">The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.</a> Current biology : CB. 10, 865-868 (2000).</li><li>Dixit, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+plus-end+tracking+by+CLIP-170+requires+EB1.">Microtubule plus-end tracking by CLIP-170 requires EB1.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 492-497 (2009).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EB1+promotes+microtubule+dynamics+by+recruiting+Sentin+in+Drosophila+cells.">EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells.</a> The Journal of cell biology. 193, 973-983 (2011).</li><li>Maurer, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EB1+accelerates+two+conformational+transitions+important+for+microtubule+maturation+and+dynamics.">EB1 accelerates two conformational transitions important for microtubule maturation and dynamics.</a> Current biology : CB. 24, 372-384 (2014).</li><li>Zanic, M., Widlund, P. O., Hyman, A. A., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergy+between+XMAP215+and+EB1+increases+microtubule+growth+rates+to+physiological+levels.">Synergy between XMAP215 and EB1 increases microtubule growth rates to physiological levels.</a> Nature cell biology. 15, 688-693 (2013).</li><li>Lowery, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+cone-specific+functions+of+XMAP215+in+restricting+microtubule+dynamics+and+promoting+axonal+outgrowth.">Growth cone-specific functions of XMAP215 in restricting microtubule dynamics and promoting axonal outgrowth.</a> Neural development. 8, 22 (2013).</li><li>Marx, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+cytoplasmic+linker-associated+protein+1+(XCLASP1)+promotes+axon+elongation+and+advance+of+pioneer+microtubules.">Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules.</a> Molecular biology of the cell. 24, 1544-1558 (2013).</li></ol>

The authors declare that they have no competing financial interests.

PlusTipTracker提供了一个简单的，图形化的用户界面，可以快速自动检测几乎所有可见的EB1-GFP彗星在一个细胞或生长锥中，彗星链接进入轨道，并计算吨参数。其它出版物都报道了相似类型的软件的设计（例如，马克思等人的标签EB1动力学在生长锥18也用于定量分析）。但是，这个软件似乎是在其四通八达的交通网络独特的，因为它是自由的Danuser实验室的网站，专门设计的开放源代?...

该方法的目的是获得关于微管（MT）的量化信息加端跟踪蛋白（+ TIP）中活的生长锥动力学。 MT +技巧是一组定位于微管9,10的加末端蛋白。他们进行了一系列的功能来调节吨动力不稳定性11，包括聚合，灾难和救援率的参数。一个很好的方法，用于分析MT动力学是跟踪+ TIP EB1，其特异性地结合到生长的MT加结束1,12的行为。 EB1是众所周知的招募其他几种蛋白不断增长的MT加端13,14，和最近被确立为MT成熟因子15，推动两吨增长和灾难频15,16。 在生长锥的MT动力学许多研究都采用手工跟踪方法来测量时间的推移1-3改变EB1-GFP的动态，EB1 localizati上到MT加-端可被用作一个标记为MT聚合。审查E​​B1-GFP的彗星为吨增长了代理的一个关键好处是，MT动力甚至可以在显著吨重叠区域进行测量。而手跟踪EB1-GFP彗星的方法提供了有用的见解MT行为1-3，这是耗时的，并且可以被偏置。此外，作为异常的生长锥的行为很可能在细胞骨架动力学分钟的变化，分析MT的仅一小部分的结果（通常是在有需要时手动跟踪）可能会错过显著信息。 因此，我们测量使用的软件包，plusTipTracker 4全球MT动力学参数，采集的高分辨率，标记EB1在培养胚胎生长锥5直播之后的图像。这个软件在Danuser实验室开发的，已被用于一些研究中的各种细胞类型6-8表征MT动力学。它是一个开源的，我们ER型，基于MATLAB软件包，包括自动检测，跟踪，可视化和分析的荧光标记+小费电影。 MT的动力的具体参数是由该软件计算出的长长的名单（见参考文献4的细节），但在生长锥的MT动态分析，最有用的参数是MT增长的轨道速度（微米/分钟），增长的轨道寿命（以秒为单位），并且增长的轨道长度（微米）。该软件可以直接从Danuser实验室网站下载（下称“软件”）。而Danuser实验室目前支持+提示跟踪分析，这是纳入到一个软件包，称为U-轨道2.0，原始的，独立的软件将继续提供一个较新的接口。两个程序之间的底层算法是相同的（至少为2014），具有接口和数据输出，仅相差。对于小MATLAB和/或计算分析experi初级用户ENCE，plusTipTracker有更多人性化的功能，包括自动统计参数输出。 在这里，我们描述了用于分析在培养的爪蟾生长锥EB1-GFP的动态图像的步骤。该协议被用在最近的一篇论文研究的MT动力17。也为表达对EB1-GFP的培养生长锥的详细说明，请参阅洛厄里等人 2012 5。虽然本文主要侧重于审查中的生长锥EB1-GFP动力学，相同的方案可以用于其他类型的细胞17。对于所有类型的细胞，帧之间的时间间隔应为0.5-2秒，最优+ TIP跟踪之间。最多为4秒的帧之间的时间间隔是可能的，但这种增加的间隔时间导致额外的跟踪误差。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">plusTipTracker software</td>
			<td style="width:116px;">Danuser Lab</td>
			<td style="width:345px;"><a href="http://lccb.hms.harvard.edu/software.html">http://lccb.hms.harvard.edu/software.html</a></td>
			<td style="width:172px;">This software may be hosted by another website in the future.&#160; If the listed site does not exist, search &#34;Danuser Lab Software&#34; on a web search engine to find the site.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Matlab software</td>
			<td style="width:116px;">Mathworks</td>
			<td><a href="http://www.mathworks.com/products/matlab/">http://www.mathworks.com/products/matlab/</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

using plustiptracker software to measure microtubule dynamics in xenopus laevis growth cones

微管（MT）的正端跟踪蛋白（+ TIPS）定位于微管的日益加端和规范的MT动力1,2。其中最知名的和广泛使用的+小技巧分析MT动力学是最终结合蛋白EB1，这一切结合不断增长的MT加末端，因此，是一个标志吨聚合1。内生长锥EB1行为的许多研究已经使用费时和偏置计算机辅助，手跟踪方法来分析单个微管1-3。我们的方法是用量化的软件包，plusTipTracker 4吨动力学全局参数，继收购高分辨率，标记EB1在培养胚胎生长锥5实时图像。这个软件是一个基于MATLAB的，开源的，用户友好的，结合了自动检测，跟踪，可视化和分析的荧光标记+小费电影包。在这里，我们提出的协议使用plusTipTracker为荧光标记+提示彗星在培养非洲爪蟾生长锥的分析。但是，此软件还可以用于在各种细胞类型6-8来描述MT动力学。

使用如下所述的提供信息的几个文件，量化+动态提示在活细胞中该软件。 函数plusTipGetTracks标识（使用图1中所示的示例设置）磁迹，然后提供关于+提示轨道参数。要查看该软件已经获得的信息，进入了在步骤2.2中创建的roi_X目录。的“壮举”文件夹包含“overlayImages”，这是一系列的图像显示检测到的彗星。检查用图像分析软件可以显示彗星检测的精度这些图像?...

Watch this Scientific Journal Video about Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones at JoVE.com

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

该基于MATLAB的，开源软件包，plusTipTracker，可用于分析图像序列的荧光标记+小费量化微管动力学。

使用plusTipTracker软件测量微管动力学<em&gt;非洲爪蟾</em&gt;生长锥

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

using-plustiptracker-software-to-measure-microtubule-dynamics-xenopus

Research

JoVE Journal

Biology

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

10.3K 次观看.  Boston College. 该基于MATLAB的，开源软件包，plusTipTracker，可用于分析图像序列的荧光标记+小费量化微管动力学。 

视频: Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

塑料嵌入和非洲爪蟾胚胎切片

科研

生物学

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

从海兔的神经细胞培养的高分辨率成像生长锥

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

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 ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.