我们感谢苏珊农民和UAB的斑马鱼研究设施的斑马鱼护理的工作人员。从药理学和毒理学部提供的启动资金资助。

注:此方案经阿拉巴马大学伯明翰分校机构动物护理和使用委员会。 1，斑马鱼繁殖和蛋收藏<ol><li>开始接触化学品的前三天，组装育种坦克分隔到不同的男性和女性。每罐半路水产养殖系统水加满。 c262/c262鱼养殖坦克，把男2例，女3例每罐由分频器分离:使用网络，TG（GFP 5xERE）转移。将盖子上的每个罐和带有日期和菌株划线标示。 </li><li>第二天早晨，删除所有的障碍和倾斜挡板在养殖池的一端创建一个浅水区。让斑马鱼交配，收集胚胎在10分钟的间隔，以确保精确的发育分期。干净的鸡蛋用E3B中，轻轻地冲洗胚胎成培养皿通过茶滤网清洗。返回成鱼为永久坦克。 </li><li>使用解剖显微镜，排序，计数，并移除任何未受精，异常或损坏胚胎。地方胚胎在培养皿中E3B中，房子在28.5°C与14时10分光:暗周期。胚体密度会影响发展。将不超过100个胚胎在一个100 x 35毫米的菜不超过50的胚胎在60×15毫米的菜。 </li><li>由24个高倍视野，用含有200μM的1 - 苯基-2 - 硫脲（PTU）E3B更换介质。 PTU防止色素形成并保持斑马鱼胚胎和幼虫透明，以提高图像清晰度。注意:集中PTU股票是危险的;使E3B + PTU解决方案时戴上手套。 </li><li>在第1天受精后（DPF），手动从用细镊子每个胚胎取出绒毛膜。绒毛膜似乎不影响雌激素的穿透力，然而除去增强图像的清晰度。也没有必要从介质除去绒毛膜。注:作为一种能够替代人工dechorionation，胚胎批，可以CHEmically dechorionated用1毫克/毫升的链霉蛋白酶处理，如前所述11。 </li></ol> 2，化学胚胎的治疗<ol><li>治疗前一天，制备贮备液的每种化学品在DMSO中1,000倍浓度（或适当的溶剂中）。例如，揭露胚胎至10微米双酚A（BPA），准备一个10毫米的BPA原液在100％DMSO。这可以确保均匀的DMSO浓度在各处理及允许的无毒1:1,000稀释的DMSO作为载体对照。店内库存解决方案在-20°C对于这个协议中，胚胎将暴露于1μM和10μMBPA，18.5 nM和1.85μM染料木黄酮，367 nM的雌二醇（阳性对照）和载体（0.1％DMSO中，阴性对照）。 注意:化学品如BPA和雌二醇是危险的，并且可能对人类胎儿的有害作用。处理内分泌干扰物小心，始终使用合适的个人防护装备。</li><li>在治疗的当天，解冻备现货的化学溶液。吹打1毫升E3B + PTU至1.5 ml离心管中，加入1微升的股票化学溶液稀释1:1,000。涡大力。二甲基亚砜稀释成1:1000 E3B +机动部队作为车辆控制。用1毫升E3B + PTU在1.5 ml离心管作为未处理的对照。 </li><li>使用大孔塑料移液管，从培养皿中转移胚胎到96孔板的各孔中，每3个胚胎井，每个条件3的孔中。为了防止在长时间的治疗蒸发，省略外部行和列的板块（行A和E，列1和12）的胚胎。外口井可以充满300微升E3B + PTU的。适当标记板盖。 </li><li>从每孔用细尖的塑料移液管取出介质。工作迅速，以避免晒出胚胎。 </li><li>加入200μl的处理溶液为对应良好。将盖子下·rneath板为指导，​​以确保除了治疗到适当的水井。每个涡管移液分发化学溶液之前。在视觉上确认每个胚是在处理液中。如果胚胎是在井的侧面，使用干净的移液管将它们洗入井的处理剂溶液。 </li><li>将板在孵化器在28.5°C。胚胎将用于荧光在72个高倍视野进行分析。 </li></ol> 3，自动在96孔板胚胎成像<ol><li>通过加入10微升4毫克/毫升的三卡因向每个孔麻醉胚胎。 注:麻醉前成像胚胎。在24-96 HPF，斑马鱼具有自发运动和惊吓反应到图像采集过程中不断变化的光源。在成像过程中麻醉胚胎减少运动。 </li><li>将96孔板中的机动阶段和校准阶段。使用蔡司禅蓝2011软件显微镜automati关于和控制，选择样品载体选项，并选择多井96。选择校准 。按照逐步指示校准阶段。 注意:这一步后不要取消选中的瓷砖按钮。如果瓷砖按钮处于未选中状态，校准设置可能会丢失和96孔板将需要重新校准。 </li><li>使用10倍物镜和明场（BF）的设定，确定阳性对照胚胎，并适当地设置曝光设置为高炉和GFP通道，确保荧光信号不饱和相机。专注于单一胚胎和编程显微镜获得3总的Z-部分，一个像50微米以上，一个50微米低于目前的焦平面。 注:由于不同的组织占据不同的焦平面，这将确保每个斑马鱼图像被捕获在焦点置于心脏和肝脏。 </li><li>设置软件参数，以获得采用GFP和BF channe平铺图像LS。根据收购选项卡中，选择高级设置 。选择瓷砖的区域，然后瓷砖。选择圆好选择。 注:平铺用于创建每一个合成图像良好。单场 - 视捕获每个只有一小部分好。平铺功能，可以自动捕捉多个相邻字段的视图从每口井，创造了整个井的合成图像。因此，能够自动捕获每板96的合成图像，其中每一个合成图像包含从一个单一的井数十个单独的图像。 </li><li>选择载体和填充因子90％。选择90％的填充系数将捕捉所选择的井，使得90％的每个孔的面积将是可见的合成图像中的多个图像。 注意:当选择的填充因子，示将显示代表要被成像的区域。选择的百分比，其中将包括井的区域为适合实验。 </li><li>在选项下，选择重叠的所需量。 注:当为一个单一的代表图像拼接在一起的瓷砖，5〜10％的重叠使得图像之间的接缝平滑。然而，如果图像拼接是不必要的，使用0％的重叠会降低成像时间。使用90％的填充因子和5％的重叠，单个井的合成图像由59个人的图像。 </li><li>启动成像。该显微镜将自动获取图像。 </li><li>手动检查每个合成图像以及组织特异性荧光（ 图1）。 </li><li>注:这是最好的开始与阳性对照，以确认该化学品接触观察实验性治疗的井前，任职。 </li></ol>胚胎在96孔板4。手动图像捕捉注:要确认的结果，使用20倍长工作距离物镜来交流QUIRE更详细的图像以手动方式（ 图2）。 <ol><li>使用20倍物镜和明场（BF）的设定，确定阳性对照胚胎，并设定为BF和GFP通道曝光设置适当，确保荧光信号不饱和相机。 </li><li>手动识别单个胚胎和采集图像。 注意:一旦曝光设置已被选定为阳性对照胚胎，不会改变设置。这使得荧光强度的比较，因为每幅图像被均匀的条件下拍摄的。 </li></ol>

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该作者有没有竞争的财务权益。

本协议描述了一个简单的方法来自动图像组织特异性的荧光斑马鱼胚胎。该协议是使用蔡司Axio上观察发展。 Z1的禅蓝2011软件，但该技术可以使用任何倒置显微镜电动载物台和显微镜控制软件，可以进行平铺，以创建复合图像调整。装备倒置显微镜具有机动阶段可以提供一个实用，更便宜的替代品购买专用设备，高内涵筛选。电动载物台的范围从几千元到数万美元，这取决于在舞台上的显微镜和质量的品牌和型号。 还有协议中的几个关键步骤。一些化学物质是光敏感的，因此，它可能是有益的治疗过程中孵育在黑暗中的胚胎。斑马鱼的胚胎是干燥敏感。因此，对于化学卡尔暴露持续一天以上，以填补沿着96孔板的边缘处的井用媒体来限制蒸发（协议步骤2.3）是至关重要的。 同样重要的是确保该阶段仔细校准（协议步骤3.2）。一个常见的​​错误是将板放置到舞台上没有完全固定板。在这种情况下，舞台上的板的位置可当移动台改变，这可能导致一些孔可以部分地或不成像的。当在连续成像几个板，也并非绝对必要重新校准阶段对于每个板，只要板是由同一制造商，都放置在阶段在相同的方向。 当设置自动图像拼接参数（协议的步骤3.5），确保广大每个井将进行成像。在3 DPF及以上，斑马鱼幼虫倾向于将自己近WEL的边缘位置升，因此，建议使用90％的填充因子，以确保每个孔的边缘将被可视化。然而，更大的填充因子的时间越长，用于图像采集。 它可以在合成图像进行拼接算法来提高分辨率。如果一个拼接算法将被执行，然后将切片参数，包括5-10％的图像重叠（协议步骤3.6）。的重叠更高，更好的拼接算法的性能。在某些情况下，有10％的重叠可以允许缝合以产生用于分析使用较高的放大倍数的物镜，它不需要进一步的可视化的信息的图像。然而，更大的重叠时间越长图像采集将采取，随着越来越多的图像将每孔抓获。 0％的重叠可以被用来减少图像获取时间，并尽量减少荧光灯曝光和光漂白。在这种情况下，使用的是20倍objecti捕获选择井的更详细的图像已经（ 图2）。 图像采集时间对本协议将取决于实验参数。使用本协议中的参数图像采集（90％的填充因子，5％的重叠，三Z-每部分拍摄的图像以及50毫秒的绿色荧光蛋白通道和1毫秒为明通道的曝光时间）需要三分钟，50秒每口井。因此，有可能筛选60孔在三个小时五十分钟。这是可行的，筛选每天180井，或一式三份60独特的化合物。 该协议允许自动成像的组织特异性的荧光，但有一些限制。宽场荧光成像缺乏在许多高内涵筛选技术的使用共焦成像的分辨率。此外，荧光定量是困难的，由于在一个良好的高变异性在各斑马鱼的方向。这可避免在成像均匀斑马鱼取向条件，例如通过使用模具或用微孔板孔中，迫使斑马鱼胚胎成均匀的取向5,10。图像获取时间限制的井，可以每天进行筛选，以180的数量。筛选每天180井是一个显著改善手册成像。然而，典型的高通量方法可筛选平均为10,000口井每一天，但需要复杂和昂贵的工作站。 这个协议代表一个实际的，较便宜的替代品，可以通过与一个倒置的宽视场荧光显微镜实验室容易地适应高含量筛选。此处所描述的成像方法允许组织特异性荧光的胚胎的分析还没有被仔细地安装或取向，从而大大提高了样品制备的速度和易用性。复合图像可以被存档​​以供参考。此协议的开发是为了让高通量筛选Øf组织特异性雌激素受体配体的化学库和环境水样。另外，该成像协议可用于筛选任何荧光报道为组织特异性活性。

转基因斑马鱼已经开发了允许活动的直接可视化在信号通路，如成纤维细胞生长因子1，视黄酸2和雌激素3，在活的胚胎。这些工具使筛选任何干扰信号传导途径的化学品（如测定变化的荧光强度），或者该调制信号以组织特异性的方式（在荧光定位变化）4化学品。自动图像捕捉提高化学屏幕吞吐量大幅5,6。屏幕在现场斑马鱼自动检测荧光往往依赖于昂贵的专门设备。所谓高内涵筛选提供了高解析度，定量成像，但在使用专门的平板阅读器配备的共聚焦显微镜7,8的成本效益。该方法的目标是自动地检测组织特异性荧光在斑马鱼胚胎在使用标准的落射荧光显微镜用96孔板中。组织特异性的荧光可以使用这种技术来区分，可能是缺乏获得专门的平板阅读器或高内涵筛选设备实验室一个合理的方法。 在这个协议中，我们使用自动成像技术来检测组织特异性雌激素受体（ER）在活斑马鱼激动剂3天受精后（DPF）。转基因品系的Tg（5xERE:GFP）c262/c262包含5个串联的雌激素反应元件的DNA序列（ERE）的绿色荧光蛋白（GFP）的上游侧3。在没有配体时，预期结果通常无效。配体结合触发的构象变化，使受体结合ERE DNA和调节转录9。 5xERE:GFP的鱼可以用来筛选ER调节剂的化学库，可用于筛选环境水样的雌激素污染物。

<table border="0" cellpadding="0" cellspacing="0" style="width:885px;" width="886">
	<colgroup>
		<col />
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:100px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:451px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic transfer pipettes; wide bore</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">13-711-23</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic transfer pipettes; fine tip</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">13-711-26</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-well, round, flat bottom plates</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">21-377-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 x 35mm paltes</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">08-757-100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 x 60mm plates</td>
			<td style="width:100px;">Fisher</td>
			<td style="width:119px;">08-757-100B</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 fine forceps</td>
			<td style="width:100px;">Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>tip dimensions 0.05 x 0.01 mm, for manually removing chorions from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tricane methanesulfonate</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:119px;">A5040-25G</td>
			<td>see Zebrafish Book for recipe (http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml63)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-phenyl 2-thiourea (PTU)</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:119px;">P7629-10G</td>
			<td>Prepare 20 mM stock (100x) and use at 200 &#956;M in E3B&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axio Observer Z1</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:451px;">Protocol requires an inverted fluorescence microscope with a motorized stage</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20x long working distance objective</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>We use an objective with 0.4 NA and 8.4 mm working distance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiocam HRm digital camera</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ZenBlue 2011 microscope control software</td>
			<td style="width:100px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:451px;">Protocol requires microscopy automation and control software to enable capturing of tiled images using a motorized stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>&#160;MB-1; 25 grams</td>
			<td>make 2% solution in RO water for use in E3B (below)</td>
		</tr>
		<tr height="273">
			<td height="273" style="height:273px;">E3B</td>
			<td style="width:100px;"></td>
			<td style="width:119px;"></td>
			<td style="width:451px;">60X E3 SOLUTION: 
			NaCl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; - 17.2 grams 
			KCl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; - 0.76 grams 
			CaCl2-2H2O&#160;&#160;&#160;&#160; - 2.9 grams 
			MgSO4-7H2O&#160; - 4.9 grams or MgSO4 &#8211; 2.39 grams 
			Dissolve in 1 liter Milli-Q water; store in sterile 1 liter bottle. 
			 
			1X E3B SOLUTION FOR ZEBRAFISH: 
			60X E3&#160;&#160;&#160;&#160;&#160; 150mls 
			2% methylene blue&#160;&#160;&#160;&#160; 100&#956;l&#160; 
			Bring to 9 liters with Milli-Q water 
			&#160;</td>
		</tr>
	</tbody>
</table>

semi-automated imaging of tissue-specific fluorescence in zebrafish embryos

斑马鱼的胚胎是一个强大的工具，用于小分子的大规模筛选。表达荧光报告蛋白的转基因斑马鱼经常被用来鉴定调节基因表达的化学品。化工屏幕，测定荧光活斑马鱼往往依赖于昂贵，专用设备高内涵筛选。我们描述了使用标准的落射荧光显微镜的过程与电动载物台自动图像斑马鱼胚胎和检测组织特异性的荧光。利用转基因斑马鱼是通过GFP的表达报道雌激素受体的活性，我们开发了一种半自动化的程序来筛选雌激素受体的配体激活的记者在一个组织特异性方式。在这段视频中，我们描述了在96孔板排列斑马鱼胚胎在24-48小时后受精（HPF），并补充说，结合雌激素受体的小分子过程。在72-96个高倍视野，每个图像以及从整个板被自动收集并手动检查组织特异性荧光。该协议表明，检测雌激素激活受体在心脏瓣膜，但不能在肝脏的能力。

图1示出的合成图像由一个96孔板的各孔中。每个复合图像由用5％的图像重叠59的单个图像。注意，这个活斑马鱼是随机取向于各孔中，但我们能够从肝脏区分荧光的心脏。明场图像作为参考来评估斑马鱼的方位和可视化形态异常（ 图1A和1C）是有用的。使用10x物镜自动成像被用于筛选化学物质，并确定哪些保证人工评估在更高的放大倍数， 图2示出了用于更详细的分析20倍长工作距离物镜所捕获的图像。这些图像的情况下直接从显微镜载物台上取下板在图1所示的自动扫描之后进行的。使用20倍的目标，在心脏的荧光可以精确定位于心脏VALVES（ 图2C-2F）。偶尔，胚胎的取向可能是次优的用于捕获图像，并可能导致模糊的结果。配售3斑马鱼每口井和表演一式三份各处理（协议的步骤2.3）提供了多种机会，准确可视化荧光。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51533/51533fig1highres.jpg" src="/files/ftp_upload/51533/51533fig1.jpg" /> 图1自动复合成像揭示了组织特异性荧光心脏瓣膜和肝脏明场（A，C）和荧光（B，DH） 的Tg的图像（5xERE:绿色荧光蛋白），与指定的化学品的第2天治疗c262/c262后的胚胎受精（DPF）和成像在3 DPF。每个面板显示单井（与5％59单​​个图像组成的合成图像从96孔板重叠）。 甲乙 ，胚培养于0.1％DMSO（载体对照）的GFP阴性。CH，胚胎孵育100纳克/毫升的17β-雌二醇（E2），1.85μM染料木素（根） ，或在心脏瓣膜（箭头）和肝（箭头）10μMBPA展览荧光，与18.5 nM的根或1μM的BPA展览荧光在心脏瓣膜，但不能肝脏处理的胚胎。 B&#39;，从盒装区域的单个幼虫在面板B。的D数字化放大的图像"，从面板的D盒装区域进行数字放大的图像，等等。比例尺= 500微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51533/51533fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51533/51533fig2highres.jpg" src="/files/ftp_upload/51533/51533fig2.jpg" /> <stron。克&gt;图2中的GFP阳性心脏瓣膜的详细可视化 的Tg（5xERE:GFP）在96孔板中暴露于化学物质表示c262/c262胚胎（参见图1）分别以较高的放大倍率手动成像，允许在GFP可视化房室瓣（箭头）和心脏的主动脉瓣（箭头）。 B，D荧光和明场图像合并等。所有图片都是侧视图，前部左侧，背侧顶端。比例尺= 50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51533/51533fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos at JoVE.com

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

这里描述的是一种协议，组织特异性荧光的斑马鱼胚胎半自动化成像。

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

semi-automated-imaging-tissue-specific-fluorescence-zebrafish

Nanyang Technological University

Research

JoVE Journal

Biology

9.5K 次观看.  University of Alabama at Birmingham. 这里描述的是一种协议，组织特异性荧光的斑马鱼胚胎半自动化成像。 

视频: Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

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

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

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

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

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

科研

生物学

Generating Chimeric Zebrafish Embryos by Transplantation

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

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

移植嵌合斑马鱼胚胎生成

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

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

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

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

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

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

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

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

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

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

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 ...

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 ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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

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

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

组织特异性荧光的斑马鱼胚胎半自动成像

摘要

探索更多视频

此视频中的章节

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

移植嵌合斑马鱼胚胎生成

半自动小红桃光学心跳分析

有丝分裂的siRNA转染后的时间推移成像

在现场的斑马鱼胚胎的基于双光子Photoactivation

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

直立成像果蝇胚胎

影像生活细胞的形状变化果蝇胚胎

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

组织特异性荧光的斑马鱼胚胎半自动成像

摘要

探索更多视频

此视频中的章节

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

移植嵌合斑马鱼胚胎生成

半自动小红桃光学心跳分析

有丝分裂的siRNA转染后的时间推移成像

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

直立成像果蝇胚胎

影像生活细胞的形状变化果蝇胚胎

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

在现场的斑马鱼胚胎的基于双光子Photoactivation