感谢由于热尼亚图图形布罗德斯基;请提供neurogenin1记者一行乌韦Strahle和维亚切斯拉Kalchenko，道格拉斯卢茨和列昂尼德Roitman两个光子uncaging的技术咨询和援助; Maayan Tahor提供技术援助和Suliman Elsadin阿莫斯Gutnick本书稿的意见。在Levkowitz实验室的研究是由德国和以色列基金会（批准数二千〇七分之一百八十三）;以色列科学基金会（批准号928/08）和哈里特马塞尔德克尔基金会的支持。 GL Tauro的职业生涯中的生物医学研究的发展主席是现任。

我们描述了一个协议使用笼荧光标记的细胞，但是，其他照片激活的染料和蛋白质可以同样使用。 1。注射笼荧光<ol><li>准备5％的股票，共轭葡聚糖4,5 - 二甲氧基- 2 - 硝基苯（DMNB）笼荧光素（10,000兆瓦右旋糖酐，阴离子，Invitrogen公司，分子探针，卡尔斯巴德，加利福尼亚的解决方案（5毫克笼荧光素/ 100μL0.2M氯化钾）猫没有D - 3310）。分装和储存在-20 ° C。注意DMNB笼荧光是对光敏感，应保持在黑暗中。 </li><li>准备注射槽"斑马鱼书"中所述的1％琼脂糖（Sigma公司，圣路易斯，密苏里州，猫没有。A9539 ）。 </li><li>准备一个100 × E3原液稀释2升的E3介质。 </li><li>在注射前的晚上，准备交配笼分离所需的转基因株系的女性表达一个可见的荧光（GFP，RFP等）用于本地化的photoactivati​​on靶细胞具有里程碑意义的（S）的男性（S）。 </li><li>准备使用微量拉马（萨特仪器，诺瓦托，CA，P - 97）的毛细管注射针头（灯丝，薄壁玻璃毛细管世界精密仪器，萨拉索塔，佛罗里达州，猫没有。TW100F - 6）。 </li><li> 5％荧光素冰笼解冻等份。 </li><li>在0.2M氯化钾笼5％荧光素原液稀释到终浓度为1％，并保持在冰上。避免光线照射。 </li><li>在第4步准备安装交配过。 </li><li>规定，尽快为他们收集受精卵。东方单细胞阶段的胚胎，在琼脂糖注塑模具 1 E3的覆盖。 </li><li>使用一个microloader提示（Eppendorf公司，德国汉堡，德国，猫没有。5242 956.003）回填〜1微升1％笼荧光素溶液注射针头。 </li><li>将装入一个显微附加气动力微量（气动picopump，世界精密仪器，萨拉索塔，佛罗里达州，PV820）。针</li><li>调整注射量为2 - 3nl和直接注入细胞的细胞质中的笼中荧光素。注入至少50％的实验胚胎。 </li><li>在黑暗中培养的胚胎在28.5 ° C在E3展到所需的发展阶段。如果胚胎受精后超过24小时以上的分析，添加0.1％苯基（PTU，Sigma公司，圣路易斯，密苏里州，猫没有。22290-9），抑制色素沉着。 </li></ol> 2。胚胎安装<ol><li>准备新鲜E2的解决方案（解决方案的一部分，可以储存在4 ° C一个星期）。 </li><li>大衣60毫米培养皿溶解在E2介质层薄薄的1％琼脂糖。 </li><li>准备2％低熔点琼脂糖（超纯水LMP琼脂糖，Invitrogen公司，卡尔斯巴德，加利福尼亚，猫没有16520100。）嵌入:琼脂糖溶于无菌双蒸水，微波炉加热至沸腾。保持该管的第加热过程中的开放。到离心管分装1毫升，并保持在一个加热块72 ° C。 </li><li>取出轻轻拉出绒毛膜除了尖锐的镊子（第5号）的胚胎chorions。琼脂涂层盘E2的解决方案中的胚胎（见步骤2）。 Dechorionated胚胎应处理火抛光玻璃巴​​斯德吸管，因为他们可能会粘到聚丙烯移液器。避免dechorionized胚胎暴露在空气中，液体的表面张力，因为这些胚胎可能损坏。 </li><li>选定的胚胎收集的E2的体积小，使用火抛光的玻璃巴斯德吸管，放在60毫米培养皿中的胚胎。安装旁边的胚胎下降了2％琼脂糖（〜150μL）（步骤3）。快速混合两滴获得最后一个同质〜1％琼脂糖混合物和使用其背方所面临的客观镜头在解剖显微镜东方胚胎。 </li><li>等待，直到琼脂糖已经凝固，直到嵌入式胚胎浸没在液体中添加E2的介质。这是因为它可能是从琼脂糖发布后的photoactivati​​on过程中，希望获得每道菜的单胚胎。 </li></ol> 3。 Photoactivati​​on <ol><li>我们使用的Zeiss LSM 510 META NLO两个光子显微镜配备宽带可调（700 - 1020nm）埋汰惠普从Spectraphysics飞秒激光。 </li><li>放在安装板固定在显微镜下的胚胎。在胚胎的荧光灯具有里程碑意义的可视化使用40x/0.8W Achroplan水浸泡目标。对于下面的步骤，我们使用带通滤波器（BP）的500 - 550nm的非退扫描探测器（NDD）。 </li><li> photoactivati​​on过程的先决条件，获得通过收购从最腹侧形象系列双光子激光860nm波长（绿色荧光蛋白检测）检测到的荧光信号的背侧部分空间感兴趣的区域的荧光信息。为此，可以得到足够的分辨率，使用彼此之间收购的焦平面跨度的6μm。 </li><li>选择目标z平面photoactivati​​on和使这一地区成为关注的焦点。根据实验的目的，感兴趣的区域，可以是内部或外部的GFP明显的地标。在z平面的单一的形象。保存图像，并保持在一个单独的窗口打开。 </li><li>设置的双光子激光波长720nm，当激光锁模打开"编辑"漂白"菜单。定义为photoactivati​​on利息率（ROI）的地区，在860nm（步骤4）使用的"定义地区"菜单图像。这是后移的激光波长为720nm，因为后者的动作变化的x，y位置所观察到的领域，选择这个图片上的投资回报率的关键。 </li><li>在"编辑漂白"菜单中photoactivati​​on的调整以下参数（见第9步）:一）相对激光功率; B）迭代次数; C）扫描速度。 </li><li>按"死神"在"编辑漂白"窗口中，等到激光停止。 </li><li>切换到860nm，获得一个单一的平面图像，以评估造成的光敏材料的强度和图像采集的第二个系列，以评估激活域的Z -跨度（厚度）。 </li><li> photoactivati​​on考虑以下参数: <ol class="lalpha"><li>一是要凭经验确定photoactivati​​on不同的两个参数的最佳条件:激光强度和持续时间的photoactivati​​on。相对激光功率可控制的声光调制器（AOM）的传输。的photoactivati​​on时间是一个迭代和扫描速度的功能。 </li><li>确定荧光强度的线性范围，以避免饱和的uncaged荧光。 </li><li>得到的荧光强度的uncaged分子和光敏克隆 2 Z轴跨度之间有一个线性相关。 </li></ol></li></ol> 4。 Uncaged荧光检测<ol><li> photoactivati​​on后，提高在黑暗中的胚胎在28.5 ° C在E2的介质含有0.1％的机动部队，以防止皮肤色素沉着的外观。 </li><li>在理想的发展阶段，在解剖显微镜下的胚胎从琼脂糖中删除。使用尖的手术刀，使"V"是对胚胎的头部指向创建一个"V"附近的胚胎在琼脂糖形切口。胚胎的头部附近的"V"点放置在一个封闭的镊子，轻轻推开分离钳沿胚胎的长度琼脂糖和释放到培养基中的胚胎。 </li><li>用火抛光的巴斯德吸管收集胚胎和修复的多聚甲醛（PFA，1 × PBS，pH值7.2的4％）在室温下是3小时或过夜，在4 ° C。 </li><li>取出固定液和冲洗至少5分钟，在室温下在PBST的2-3倍。 </li><li> 100％甲醇10分钟洗净的胚胎，在-20 ° C的新鲜甲醇和孵化补充至少2小时。 </li><li>补充水分（各5分钟）在一系列PBSTr甲醇稀释连续孵化的胚胎:80％，60％，40％和20％。洗净PBSTr 2 × 10分钟。 </li><li> 30分钟，在室温下孵育500μL封闭液。 </li><li>加入抗荧光素 - 碱性磷酸酶（AP）的Fab片段（罗氏，帕洛阿尔托，加利福尼亚，猫。。11426338910），在封闭液稀释1:500，孵育4℃过夜° C（在其两侧离心管） 。 </li><li>在PBST洗5 × 15分钟。 </li><li>洗5分钟，在TNT炸药在0.2M氯化钠。 </li><li>在TNT的清洗5分钟0.4M氯化钠。 </li><li> Prestain解决方案新鲜配制平衡的3 × 5分钟。 </li><li>一个红色片剂（罗氏，帕洛阿尔托，加利福尼亚，猫。11496549001）2毫升Prestain溶液溶解。这是推荐使用的Whatman 2号文件，省略未溶解颗粒过滤解决方案。另外，降速的解决方案和使用明确上清液。 </li><li>添加500μL，每管的快速红溶液，在室温下孵育在黑暗中，和显示器色彩的发展，直到达到所需的信号 - 背景比（20分钟到几个小时）。如果后台的发展，转移到4 ° C。孵化一小时后，一个新的快速红染色液补充。 </li><li>在PBST洗3 × 5分钟停止染色反应。 </li><li> 4％PFA固定在室温为20分钟。 </li><li> PBST洗2 × 5分钟。 </li><li>通过一系列的25％，50％和75％甘油的PBS清除胚胎直到胚胎落户在试管底部。了几天的胚胎可以保存在4 ° C。 </li></ol> 5。 Uncaged区的荧光可视化<ol><li>准备安装在显微镜幻灯片:使用1 mL注射器适用于四个点，在幻灯片上清晰的硅脂，放置在彼此的距离〜15毫米。安装cente胚胎r在〜200μL75％的甘油和盖玻片（0号; 18x18mm）的顶部，轻轻一推，对硅胶甘油溶液，直到占据幻灯片之间的表面。 </li><li>广场走上舞台安装胚胎，并把它使用到40倍的目标重点。收购共聚焦图像，使用488nm氩和543nm氦氖激光器作为激发源的Z系列。 </li><li> photoactivatated域的Z轴跨度可以使用三维重建uncaged使用图像分析软件的胚胎进行分析。 </li></ol> 6。解决方案<table border="1"><tbody><tr><td> 100 x E3 </td><td> 174mM氯化钠，氯化钾21MM，12MM硫酸镁4，18MM的Ca（NO3），15MM的HEPES，pH = 7.4的。消毒过滤和储存在4 ° C </td></tr><tr><td> 20 x E2 </td><td> 0.3M氯化钠，氯化钾10MM，20MM氯化钙2 * 2HOH 硫酸镁 ，20MM * 7HOH，3MM KH 2 PO 4，0.8mm的NA 2 HPO 4 * 2HOH，滤液在室温下存储</td></tr><tr><td> 1 x E2 </td><td>双蒸水稀释20 × E2和添加新鲜的碳酸氢钠3（终浓度为0.7mm），青霉素和链霉素（稀释笔链球菌解决方案的终浓度为1 ×，其中含有青霉素和链霉素每毫升100毫克（Invitrogen公司100U卡尔斯巴德，CA，猫没有。15140-122）） </td></tr><tr><td> PBST </td><td> 0.1％吐温20的PBS </td></tr><tr><td> PBSTr </td><td> 0.3％的Triton的PBS </td></tr><tr><td>封闭液</td><td> 10％BSA，1％，0.3％的Triton的PBS二甲基亚砜</td></tr><tr><td> TNT炸药</td><td> 100MM的Tris - HCl pH值7.5，150mm的氯化钠，0.5％Tween - 20的双蒸水</td></tr><tr><td> Prestain解决方案</td><td> 100MM的Tris - HCl pH值8.2，0.4M氯化钠，0.1％Tween - 20的双蒸水</td></tr></tbody></table> 7。代表性的成果例如一个双光子显微镜的使用photoactivate生活在一个斑马鱼胚胎的代理商。使用类似的方法，我们以前追溯到在斑马鱼脑 2,3的光活化标记的神经祖细胞谱系。图1显示了笼前携带neurog1的斑马鱼胚胎神经板的荧光标记的示踪染料photoactivation::GFP转基因记者，作为一个生活的内在具有里程碑意义的。 胚胎注射笼荧光素在细胞阶段和琼脂糖嵌入在萌芽阶段。在3 - 5体节期的空间坐标测量的neurog1:GFP转基因胚胎uncaging（图1A，A"），以确定的感兴趣区域（ROI）。我们uncaged在一个特定的神经板（图1B，B&#39;），域的荧光谱系示踪。随后，这个标记的神经祖子域的命运是确定prim5阶段（受精后24小时）uncaged荧光（图1C，C"）的免疫。 双光子激光uncaging的程度，即uncaged荧光的强度和厚度的光敏域（Z -跨度），可以通过调整两个参数来控制激光强度和持续时间。后者是可以控制的迭代次数和扫描速度的调整（见第3部分，第9步; Russek百隆，2009）。在图1所示的例子中，我们使用12％AOM的传输相对激光功率，20次迭代25.6微秒/像素扫描速度。使用这些设置，我们标记一个包含9-10细胞的30​​μm的一个光敏Z -跨度（1-2单元格行）的投资回报率。 <img src="/files/ftp_upload/1902/1902fig1.jpg" alt="图1" /> 图1。一个代表在现场使用双光子显微镜的斑马鱼胚胎的笼荧光的photoactivation结果。 示意图（AC）和代表图像的photoactivati​​on过程中（A&#39; - C"）。现场斑马鱼笼荧光示踪染料注入1细胞阶段的胚胎（A，A"）表达GFP神经元素- 1启动子的控制下（neurog1::GFP）。在3-5 - 体节期，前脑原基（间脑）的离散面积光敏uncaged荧光示踪染料可以检测到（B，B&#39;）。 photoactivati​​on程序后，胚胎培养在28.5 ° C和脑细胞中含有uncaged荧光抗荧光免疫追溯到在受精后24小时（HPF，C，C"）。电话奠，间脑，端脑。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+caged+neurotransmitters.">Using caged neurotransmitters.</a> Nature. 337, 583-584 (1989).</li><li>Zucker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photorelease+techniques+for+raising+or+lowering+intracellular+Ca2.">Photorelease techniques for raising or lowering intracellular Ca2.</a> Methods Cell Biol. 40, 31-63 (1994).</li><li>Neveu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+caged+retinoic+acid+for+one-+and+two-photon+excitation+in+zebrafish+embryos.">A caged retinoic acid for one- and two-photon excitation in zebrafish embryos.</a> Angew Chem Int Ed Engl. 47, 3744-3746 (2008).</li><li>Shestopalov, I. A., Sinha, S., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-controlled+gene+silencing+in+zebrafish+embryos.">Light-controlled gene silencing in zebrafish embryos.</a> Nat Chem Biol. 3, 650-651 (2007).</li><li>Ando, H., Furuta, T., Tsien, R. Y., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-mediated+gene+activation+using+caged+RNA/DNA+in+zebrafish+embryos.">Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos.</a> Nat Genet. 28, 317-325 (2001).</li></ol>

光激活的化合物分子，其功能被屏蔽，直到它们与特定波长（通常是紫外线）诱导光化学反应转换成一种生物或化学活性状态的分子，照亮。这些探头在细胞生物学研究提供了非常强大的工具，因为激活可精确控制时间和空间限制了其暴露在光线下。 多光子显微镜的显着优点是比较深的光学穿透和减少非特异性光毒性。激活过程中，必须吸收低能量的两个光子的光激活化合物，在同一时间。由于此类事件的概率是依赖于光子密度，激活焦平面仍然受到限制。激活区域，因此可以选择性地操纵在一个组织内定义的卷。 我们提出了一个协议笼使用双光子显微镜，在活的斑马鱼胚胎荧光photoactivati​​on。在具体的例子所示，在此我们使用双光子显微镜诱导笼荧光素光解，为了纪念在早期胚胎阶段的细胞，并在其后跟踪标记在发展中国家斑马鱼脑细胞谱系。相比激光闪光灯，在活化示踪染料结果在细胞和缺乏解决几十Z轴4,5，双光子基于photoactivation photoactivation可以达到的空间分辨率一个获得一个近端一到两个单元格行2,3的轴向分辨率的几微米。 程序报告本可以利用多种化合物在单细胞活体标本决议的活化。例如，枫蛋白同样可以使用标签的细胞，其从绿色到红色fluorescenct 蛋白 6,7的光转化。光激活蛋白，可应用于包括光门控的离子通道，Channelrhodopsin，可调节神经元的活动和光敏剂，如KillerRed，从而引起细胞死亡后，光线的照射下9。 已报道的各种笼分子，允许通过操纵细胞外和细胞内的化合物10的生理过程的调节。这些措施包括，积极的神经递质（如谷氨酸 11,12）和第二信使（如钙 13）和类固醇激素（如视黄酸 14） 。最后，在斑马鱼的基因激活和沉默的照片介导的控制已经出台。笼的反义寡核苷酸（morpholinos）和RNA分子的双光子基于photoactivation可用于基因敲除和增益功能，在一个活的斑马鱼胚胎15,16限制细胞群。 总而言之，基于双光子活的斑马鱼的胚胎照片激活是:1）精确的沿X，Y，Z轴的两个单元格行。 2）定量的photoactivati​​on程度是可以控制的。 3）通用性，可应用于各种照片兑换蛋白。

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Dextran-conjugated 4,5-dimethoxy-2-nitrobenzyl (DMNB) caged fluorescein (10,000 MW dextran, anionic)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D-3310</td>
<td>molecular probes</td>
</tr>
<tr>
<td>Agarose for injection trough and coated plates</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9539</td>
<td>&nbsp;</td>
<tr>
<td>Thin Wall Glass Capillaries with filament</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-6</td>
<td>&nbsp;</td>
<tr>
<td>Micropipette puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Microloader tip</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5242 956.003</td>
<td>&nbsp;</td>
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<td>Pneumatic picopump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PV820</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>22290-9</td>
<td>&nbsp;</td>
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<td>Low melting point agarose for embryo mounting</td>
<td>&nbsp;</td>
<td>Ultra Pure LMP agarose</td>
<td>16520100</td>
<td>&nbsp;</td>
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<td>Anti-Fluorescein- alkaline phosphatase (AP) Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426338910</td>
<td>&nbsp;</td>
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<td>Fast Red</td>
<td>&nbsp;</td>
<td>Roche</td>
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two-photon-based photoactivation in live zebrafish embryos

Photoactivati​​on一个活的有机体中的目标化合物已被证明是一个有价值的方法，调查各种生物，如胚胎发育，细胞信号和成人的生理过程。在这方面，多光子显微镜的使用，使深部组织的一个给定的光反应剂在单细胞分辨率定量photoactivati​​on。由于斑马鱼的胚胎是透明的，其发展可以监测体内。这些特点使斑马鱼的一个完美的模式生物，用于控制各种化学制剂和聚焦光线的蛋白质的活动。在这里，我们描述双光子显微镜的使用，以诱导荧光，化学笼激活，从而允许我们按照活的斑马鱼胚胎细胞的命运。我们使用表达一个活的遗传具有里程碑意义的（GFP）的定位和精确瞄准任何利益细胞的胚胎。此过程同样可以用于精确的光诱导活化蛋白质，激素类，小分子和其他笼化合物。

Watch this Scientific Journal Video about Two-Photon-Based Photoactivation in Live Zebrafish Embryos at JoVE.com

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

多光子显微镜允许控制与深厚的光学渗透和减少光毒性低能量的光子。我们描述了使用这项技术在斑马鱼胚胎活细胞标记。该协议可以很容易地适应各种光反应的分子的光感应。

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 ...

two-photon-based-photoactivation-in-live-zebrafish-embryos

Nanyang Technological University

Research

JoVE Journal

Biology

11.5K 次观看.  Weizmann Institute of Science. 多光子显微镜允许控制与深厚的光学渗透和减少光毒性低能量的光子。我们描述了使用这项技术在斑马鱼胚胎活细胞标记。该协议可以很容易地适应各种光反应的分子的光感应。

视频: Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

科研

生物学

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

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.  ...

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 ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

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 ...

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.

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

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. ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

在现场的斑马鱼胚胎的基于双光子Photoactivation