Name: culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy
Uploaded: 2017-02-24T15:00:00.000+00:00
Duration: 11 min 55 s
Description: Watch this Scientific Journal Video about Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy at JoVE.com

我们赞赏威廉·阿彻和巴黎圣母院综合成像设备提供的支持。特别感谢被定向到Freimann生命科学技术人员为他们的持续帮助和他们的照顾和斑马鱼的养殖。这项研究是由美国国立卫生研究院，从圣母大学的国立眼科研究所DRH（R01-EY018417，R01-EY024519）和中心斑马鱼研究，巴黎圣母院，赠款支持。

注:斑马鱼中提出并维持在Freimann生命科学中心在巴黎圣母院斑马鱼设施。在这个手稿中描述的方法是通过巴黎圣母院动物护理和使用委员会批准的大学，并符合在视觉与眼科视觉研究中使用动物的研究协会的声明。 1.解决方案<ol><li>制备70％的乙醇消毒的组织培养罩和任何设备/被转移到组织培养罩试剂。 </li><li>添加2mL的2-苯氧基乙醇的1升系统水（1:500 2-苯氧基乙醇）。 </li><li>制备0.1mM的碳酸氢钠 ，pH值8.0。使用一个10毫升的注射器和一个0.2微米的孔径的注射器过滤器进行消毒在无菌组织培养罩的溶液。 注: 碳酸氢钠被用于有效地分布在fluorodishes的盖玻片表面的细胞和组织粘合剂（见步骤3.2）。 </li><li>制备的1.0M的CaCl 2和1.0M的MgCl 2。用10毫升的注射器和0.2微米孔径的注射器过滤器在无菌组织培养罩消毒。 </li><li>为了制备Hank&#39;s平衡盐溶液（HBSS）中，在每1 1mM的终浓度添加无菌的CaCl 2和无菌的MgCl 2至1×HBSS不含Ca 2+ / Mg 2+离子 ，无酚红。在无菌的环境中工作。 </li><li>为了准备培养液，混合50％1X最低基本培养基（MEM）无酚红，25％含有氯化钙和氯化镁 HBSS（参见1.4节），25％马血清（HS），10单位/ ml青霉素，和10微克/毫升链霉素。在无菌的环境中工作。 注:避免含有酚红培养基，因为它autofluoresces，从而影响信噪比。 23 </li><li>制备10毫升在1×MEM 1％低熔点琼脂糖的无酚红。熔融琼脂糖/ 1X MEM一个microwAVE。制备琼脂糖（ 例如，10毫升），为重复的再加热的小体积将发生变化，由于流体蒸发离子浓度。 </li><li>以培养前，高压灭菌1.5毫升离心管。 </li></ol> 2.光损伤范式<ol><li>暗适应<ol><li>放置2 - 3 的Tg [GFAP:nGFP] mi2004斑马鱼（或其它感兴趣的转基因斑马鱼）6 - 14月龄的成用于14天缺乏轻的环境。对于进一步的细节见参考文献2，6，24，25。 </li></ol></li><li>在系统中的水可释放2800勒克斯2，6，24，25的光日光灯二支3之间的暗适应转基因斑马鱼-含2坦克的位置。 </l我&gt; <li>斑马鱼暴露恒强光35小时。在曝光，保证水温维持31之间 - 33°C。 </li></ol> 3.准备培养（视网膜隔离的日子） <ol><li>用70％的乙醇，灭菌组织培养罩和部件/被转移到组织培养罩的工具（ 例如，含有MEM和HBSS，移液器，无菌吸头， 等等烧瓶）。 </li><li> fluorodishes的制备<ol><li>涂布一层FluoroDish中用于细胞和组织粘合剂视网膜每一个（见材料表 ）。 </li><li>稀释在0.1毫米的NaHCO 3中的细胞和组织粘合剂至70微克/毫升的终浓度，加入50μL的每个FluoroDish中，并用200微升枪头传播穿过FluoroDish中的中心的溶液（约1 - 1.2厘米直径的）。 </li><li>孵育涂fluorodishes 1 - 在室温3小时（或者，孵育O / N在4°C）。 </li><li>除去溶液，并用500μL的1×MEM的每次洗涤漂洗3次。为了避免干燥涂层fluorodishes，维护他们MEM直到视网膜外植体安装。 </li></ol></li><li>如步骤1.6说明准备培养基。培养基可存放在4℃长达1周℃。 </li></ol> 4.视网膜外植体的分离和培养注意:下面概述的协议是用于背侧视网膜，这是主要是由所描述的光损伤范例损伤视网膜区域的隔离。因此，损伤诱导的增殖和interkinetic核迁移相关的事件发生在视网膜背。然而，分离步骤可以调节根据研究者/研究问题的具体要求，以产生视网膜的区域。 <ol><li>安乐死一盏灯损坏的转基因斑马鱼一た时间在1:500 2-苯氧基乙醇。 </li><li>从一个FluoroDish中与1000微升吸管除去MEM使得仅流体遗体的薄膜。 </li><li>转移斑马鱼到干纸巾，具有弯曲一对杜蒙钳（钳子＃5，45°角）的去除眼睛，并将其转移到FluoroDish中。 </li><li>利用体视显微镜，东方与眸到FluoroDish中的盖玻片使眼睛与视神经背面是可见的（ 图1D）。 </li><li>有一对麦弗逊式Vannas剪刀除去视神经，切割靠近眼睛的后部。此外，除去结缔组织衬眼睛的外部。 </li><li>持其鼻和颞侧之间的眼用一对＃5钳子而用通过筛板1剪刀刀片刺入并沿着鼻和眼睛的颞两侧切割使得在视柄切口（ 见图1E ，分段李NE）。 </li><li>使用两对的＃5镊子，一个用于保持视网膜的背侧，另一对向腹侧从背视网膜分离，拉组织。 注意:这是可取使得透镜和神经节细胞层面临盖玻片而巩膜朝上定向背侧视网膜。 </li><li>带有一对＃5镊子除去从背视网膜巩膜，同时保持连接到视网膜与第二对＃5镊子（ 图1H，Ⅰ）的透镜。 </li><li>若要取下镜头，使用麦弗逊式Vannas剪刀切镜头后面不破坏视网膜（ 图1I，J）。有时，透镜在步骤4.7中分离。在这种情况下，通过用第二对＃5钳子保持在视网膜在切割边缘仔细移除巩膜用钳子。 </li><li>除去玻璃体而不会损坏视网膜取下镜头。弄平朝向盖玻片的神经节细胞层中的视网膜所述FluoroDish中的（ 图1L中，M）。 </li><li>围绕用1％低熔点琼脂糖10μL的视网膜和让琼脂糖固化。 </li><li>手表液体琼脂糖不解除视网膜，因为即使有轻微的升高可能会影响到重点深入到组织的能力。如果观察到吊装，用吸管去除琼脂糖。尝试添加更多的之前，让残留的琼脂糖集。 </li><li>重复步骤4.12几次加入1％低熔点琼脂糖覆盖整个FluoroDish中之前。一旦琼脂糖凝固，加入1.5毫升的培养基。 </li><li>保持在大约12小时设定在32℃的5％CO 2 /空气环境中的视网膜外植体培养，以允许视网膜从由隔离过程产生的应力恢复。设定温度为32℃，以保持在相同的温度下的光处理的斑马鱼视网膜（参见步骤2.3）。 </li></ol> 5.多光子显微镜材料表 ），40倍的Apo长距离水浸泡的目的（NA 1.15）多光子显微镜，检流计扫描仪和进行优化环境室，其中包含四张35 25mm培养皿的插入物。的图像与非退扫描检测器（R-NDD）获得的。 <ol><li>成像之前，平衡的环境室，以达到5％的CO 2 /空气的气氛。确保空培养皿插入保持器，以避免气体的漏入房间。 </li><li>打开显微镜系统。 </li><li>一旦环境室平衡，增加折射率液体到40X阿波长距离水浸泡的目的（NA 1.15）。 注意:与水相似的光学性质的折射率液体被用来避免长期成像期间的水蒸发。 </li><li>地方感视网膜植入腔室orodishes。使用明场光，将试样放置到光路和使背视网膜中区成为焦点的平面。 </li><li>使用GFP啶光集中于GFAP:nGFP阳性穆勒神经胶质细胞的细胞核（ 图2A，C）。 注:如果植体未安装平面或外植下琼脂糖积累，这将是困难的聚焦在GFAP:nGFP阳性核或他们将朦胧荧光。 <ol><li>检查是否移动到不同的区域相同的视网膜内植克服聚焦问题。否则移动到不同的视网膜外植体。 </li></ol></li><li>在图像采集软件，打开"A1 MP界面"中，"TiPad"中，"A1紧凑的GUI"和"ND采集"窗口。对于多光子成像，确保"IR NDD"选项中选择的是"A1紧凑型图形用户界面"。 </li><li>在&#39;设置搜索G&#39;字段中，选择IR-DM为1 日分色镜，并选择带通滤波器五十零分之五百二十五获得绿色荧光。 </li><li>交换机上标有"A1MP GUI"窗口中的红外激光。这将需要几分钟的激光做好准备。波长设置为910 nm的激发GFP荧光，并通过点击"A1 MP GUI"窗口中的"自动对齐"按钮调整激光。 </li><li>确保室和装备灯被关闭或在"A1 MP GUI&#39;打开快门，以避免光电倍增管的过度曝光之前覆盖。为了降低噪音水平，安置在黑暗的环境显微镜。 </li><li>获得的视图的300×300像素，场的图像在两变焦和4.8微秒/像素的像素住宅时间。大致由"A1MP GUI"，并在"A1紧凑型GUI"窗口中的增益改变了"收购区"设立了激光功率。 </li><li>设置的Z轴堆叠<ol><li>聚焦神经节细胞层上设置ND采集"窗口"的内Z&#39;-子窗口"中的z堆栈顶部焦平面。一些GFAP:nGFP阳性细胞通常位于神经节细胞层，这有助于识别视网膜（ 图2A中的D）的基极限。 </li><li>通过ONL的水平移动焦平面（ 图2A，B），其特点是模糊地标记GFAP的存在:圆形和扩大相对对口的INL（ 图2A，C）是nGFP阳性细胞。 </li><li>设置该平面的z堆栈的底部。确保整个ONL将被成像（ 图2A，B）。 </li><li>而当经历焦平面的变化在成像时期，需要重新调整，在"Z"子窗口的中间位置双击将其指定为"家"的位置。切换到"对称模式下定义"单击"相对"。 </li><li> 0.7之间的z步长设置为1微米。 </li></ol></li><li> Z-强度校正: <ol><li>应用Z-强度修正，以补偿像素亮度的损失，由于光散射时，在组织深层成像。 </li><li>要设置校正，打开"Z-强度校正"窗口。要设置"Z-堆栈范围"，选择"从ND"。 </li><li>在&#39;的z强度校正&#39;窗口（在&#39;A1MP GUI&#39;采集区域"）和增益（A1紧凑的GUI点击底部焦平面（在这种情况下，对应于神经节细胞层），并设置激光强度）。 </li><li>点击"Z-强度校正"窗口旁边的"Z值"的箭头，以确认在为所选焦平面&#39;Z-强度校正"窗口下的"设备设置"随后显示的设置。 </li><li>重复这个过程中间第二顶面，增加了激光功率和增益。附加焦平面可以根据需要添加。请参阅表1对图2的实验特定的激光和增益设置- 4。 </li><li>坐落在"A1 MP GUI"窗口中的"购置面积"。避免选择采集面积大于15，并在成像规避漂白的开始的增益高于126和增加的噪声水平。 注:由于激光和光电倍增管显微镜系统，测试激光增益设置有所不同，以获得成立，同时避免漂白显微镜最佳成像条件。 </li><li>在"Z-强度校正"窗口中，选择"相对强度校正"。 </li></ol></li><li>在"ND收购"窗口中的"时间序列"子窗口，设置持续时间8小时，间隔以"无延迟"。然后，请务必点击T中的"运行Z-校正"在"ND收购"窗口按钮，他"Z堆栈"子窗口收购3-D时间序列。 </li><li> 29°C在整个图像采集的时间 - 在27的温度维持视网膜外植体。 </li><li>整个图像采集期间，如有必要，重新调节功率水平和增益，以维持对成像后分析图像质量。对于调整执行步骤5.11.2 - 5.11.4。 </li><li>如果焦平面变化，暂停或停止运行，并再次执行步骤5.11。 注意:如果&#39;相对z校正"之所以选择在步骤5.12.7并进行一步5.11.4它不应该是必要的调整功率和增益水平，除非发生大规模漂白。 </li></ol> 6.速度分析<ol><li>提取时间，设定在图像上"感兴趣区域"。使用"时间测量"工具和时间值导出到电子表格。 </li><li>裁剪包含二的区域viding GFAP:nGFP阳性核。 </li><li>选择裁剪的区域，使得它至少包含一个核，这并不以设置参考点随后测量出分割核相对于基底的INL迁移的距离进行INM。注意:要选择留在基底INL核，它有助于准备时间序列的三维重建。 </li><li>或者，如果一个GFAP:在ONL观察整个采集期间nGFP阳性细胞，可使用固定长度从ONL细胞核到基底INL跨越的垂直线，以便确定一个参考点。 </li><li>使用"显示切片视图"功能，生成正交投影。随后，从改变&#39;片&#39;到&#39;最大强度投影"的模式。 </li><li>关闭正交投影最大的"XY"视图。取决于在该迁移/除以核最好是可见的，一个方向LSO关闭无论是"xz&#39;-或"yz&#39;视图。 </li><li>单击其余图像上用鼠标右键，并提取创建一个从这个观点新文档"功能的"XZ"或"与yz&#39;映像系列"。如果需要的话，旋转图像。 </li><li>在保留在基底INL和不经历INM核的底部水平使用&#39;手工测量"功能，绘制在图像的水平线（=基准点; 图4A - E，红色水平线）。 </li><li>测量基准线和所述转移核，使用在分析软件的"在线测量"工具（ 见图4A - E）的时间序列的基点之间的距离。 </li><li>一旦核膜分解，测量胞体的基底位置，如果它是可识别的GFP扩散到整个细胞以下。 </li><li>使用电子表格软件，绘制距离NUcleus迁移针对传递（ 图4F）的时间。 <ol><li>要确定心尖移动速度（V A），图的距离走过了核膜破裂前的时期出现（ 图4G）。 </li><li>选择图表中的数据系列，右击鼠标并插入一个线性回归曲线，包括相应的函数&#39;表达式y = mx + C&#39;。在函数的斜率的"m"代表速度v 一个 （ 图4G）。 </li><li>快速基底迁移的第一阶段重复步骤6.11.1和6.11.2来确定基底的移动速度，V B（ 图4H）。 </li></ol></li></ol>

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研究调查执政损坏的成年斑马鱼视网膜主要用于免疫组化方法5，25，26，27，28，29，30的再生机制。建立培养视网膜外植体的条件和对现象，如INM进行活细胞成像，为我们提供了一种技术，获得深入的空间和时间信息。这种技术允许确定所述偏移速度，有丝分裂和长度和细胞分裂的位置的前期期间的时机和核膜破裂的位置。此外，有可能建立在考虑到其后续的位置的分割平面与所产生的子细胞的命运。最终，这个强大的方法将确定是否中号2;米勒胶质细胞和神经元祖细胞表现不同视网膜再生过程中考虑到INM。 ，在再生反应的不同阶段确定在再生视网膜增殖细胞使用转基因斑马鱼线将帮助解密差动行为6。在这里使用的多光子显微镜的情况下，GFP报告斑马鱼线被优选作为红色荧光蛋白（RFP）的激发效率不高;然而，其他的多光子系统可以允许RFP或等效荧光团的高效率激发。 一项所述的视网膜分离过程中的最重要的步骤是安装的视网膜。视网膜必须被安装尽可能平坦的多光子光能够传递到ONL的更深的z的水平。视网膜特别是在边缘区域中，残留的玻璃体，和/或琼脂糖的积累视网膜培养下，它被用来以固定的弯曲组织中，最常见的介绍盖玻片和视网膜文化的额外空间。此外，增加的激光功率和增益必须被用于获取图像，如果视网膜不是平坦的，这将进而导致增加的光漂白和降低了视网膜培养物生存力。不正确的安装往往也导致较差的图像质量由于减少光渗透和影响来测量核已迁移，因此迁移速度的计算的距离的能力。 mi2004斑马鱼视网膜外植体:心尖迁移核膜破裂前，基础迁移的速度能够可靠地在图像序列从Tg为nGFP GFAP]确定。然而，它是目前无法确定在该转基因斑马鱼线作为整个细胞质中的GFP重新分配核膜破裂后的染色质的心尖迁移的速度。 Labeli与核染料Hoechst的视网膜外植体纳克导致在昏暗的摄取到所需的波长的变化到830纳米（Lahne＆海德，未发表的数据），这影响了组织的生存力，并造成细胞周期的停滞米勒胶质细胞核（ Lahne＆海德，未发表的数据）。期间视网膜发育，Tg为[h2afva:h2afva-GFP]为表达GFP融合于蛋白2 kca6斑马鱼已经被成功地用于监测INM，并在细胞周期的12，31的不同阶段测定的迁移速度。在未来，从Tg为[h2afva:h2afva-GFP]视网膜植kca6斑马鱼还将使染色质的整个细胞周期的可视化，并允许在成人再生斑马鱼视网膜核膜破裂后根尖偏移速度的分析。 在确定监测INM的条件在视网膜植活细胞成像从光受损成年斑马鱼和速度的相应的分析将形成基础研究理事INM的机制。一些研究开始审查INM的机制在成人使用免疫细胞化学来确定有丝分裂磷酸化组蛋白3阳性细胞核6,7的位置再生视网膜。然而，随着信号通路干扰可能无法呈现有丝分裂的位置，但可能会影响到其他参数，例如速度，有丝分裂和分裂的定时，将是不可能的/很难通过免疫细胞化学来辨别。先前，已显示错误定位，以在dynactin突变体和morphants 11显影视网膜更基底的位置是磷酸化组蛋白3阳性有丝分裂核。然而，随后的活细胞成像显示，核迁移dynactin受损的细胞被推迟细胞分裂之前一样，而增加的顶端偏移速度使细胞核到达，并在顶表面11,12经历细胞分裂。这个例子体现的活细胞成像的权力。同样，解开促进INM在成人再生斑马鱼视网膜的机制时，活细胞成像研究将补充，并在各种情况将超过，免疫细胞化学方法是有利的。 如上所述，活细胞成像提供超过免疫/静态方法许多优点;然而，它必须牢记，视网膜外植体的体外模型。为隔离过程期间发生这样的，损坏和/或培养条件可能影响细胞过程。此外，在成像过程本身可施加可能影响细胞行为23,32毒性作用。虽然有disadvantages活细胞培养的视网膜成像，它是目前获得INM的动态信息我们的最好的方法。重要的是，视网膜外植体的活细胞成像过程将适用于视网膜再生期间其他生物的问题。基于免疫的数据，它以前被认为米勒的神经胶质细胞吞噬垂死的感光器/杂物下面光致光感受器的损伤33。如果垂死的光感受器细胞吞噬功能的斑马鱼再生视网膜发生，并且可以用来研究的基本机制调整参数，以图像这个过程Live将验证。具有显示图像可以在杆细胞核和杆内的段的级别中的Tg获取丙基:Eco.NfsB-EGFP] nt19斑马鱼，应该调整条件以图像感光体吞噬作用是可行的。同样的，知识是有限的小胶质细胞方面的动态行为和有关其机制功能在退化和视网膜再生34,35。在用活细胞成像一起利用特定的小胶质细胞转基因斑马鱼也将增加我们在成年小胶质细胞再生斑马鱼视网膜36，37的功能的理解。总结，视网膜外植体活细胞成像是获得细胞过程的动态信息，并确定在再生视网膜不同细胞类型的行为和功能的有力工具。

与人类不同，斑马鱼（Danio rerio）显示出在视网膜神经元1，2，3，4的细胞死亡的健壮再生反应。肿瘤坏死因子α，即从濒死的视网膜神经元释放的信号传导分子诱导驻留在基底内核层的视网膜（INL）在米勒胶质细胞，增殖5并产生神经元祖细胞继续分化成神经元细胞之前增殖即死2种，3，4。在再生反应的增殖期，米勒胶质细胞的细胞核和其衍生的神经元祖细胞阶段进行的重复洄游图案与细胞周期（Interkinetic核迁移，INM）6 </sup<html> &gt;，7。定位在基底INL细胞核迁移到外核层（ONL），他们划分而产生的核基部返回到INL之前之前复制它们的DNA。这个过程的神经上皮发育期间利用组织学方法首先描述，而活细胞成像的方法后来被绍尔8，9，10，11，12证实了解释。两个组织化学和活细胞成像的方法已被用来确定底层INM及其发展神经上皮包括视网膜9，11，12，13的功能的机制。然而，管理在成人视网膜再生的INM机制尚未研究了很多细节外部参照"&gt; 6，7，活细胞成像将推动我们在成人视网膜再生控制INM的信号通路知识的宝贵途径。 直到最近，在视网膜INM的活细胞成像限于任一活斑马鱼的胚胎或胚胎鸡或产后小鼠视网膜植9，10，11，12，14，15，16。而从各种物种的成年动物，包括小鼠，大鼠和斑马鱼视网膜植已被用于不同的细胞生物学方法17，18，19，20，利用视网膜植公顷活细胞成像实验已经被限制向大家介绍的时间段，并没有被数小时21,22连续执行。在这里，我们描述了一个详细的协议，以文化的光损伤成年斑马鱼视网膜利用多光子显微镜6进行活细胞成像实验监测INM。调查控制INM机制时，作为INM的动态， 例如，速度可能会受到影响，而不是有丝分裂的位置，这将潜在地不使用免疫细胞化学来检测活细胞成像的方法是通过免疫组织化学方法是有利的。 在将来，这种方法有也可能被修改，以研究视网膜再生期间其它动态过程，如垂死由米勒胶质或小胶质细胞的行为的光感受器的吞噬作用。

<table><tbody><tr><td>Dumont forceps #5</td><td>World precision instruments</td><td>14098</td><td></td></tr><tr><td>Dumont forceps #5, 45&amp;deg; angle</td><td>World precision instruments</td><td>14101</td><td></td></tr><tr><td>McPherson-Vannas scissors</td><td>World precision instruments</td><td>501233</td><td></td></tr><tr><td>Fluordishes</td><td>World precision instruments</td><td>FD35-100</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon&amp;nbsp;</td><td>SMZ-1B</td><td>similar type of dissection stereomicroscope will work</td></tr><tr><td>Biological Safety Cabinet class type A2</td><td>Labconco</td><td></td><td>equivalent type will work</td></tr><tr><td>tissue culture incubator</td><td>Thermoscientific</td><td>HEPA-class 100</td><td>equivalent type will work</td></tr><tr><td>Sylvania fluorescent lamps OSFP5835HOECO</td><td>Bulbtronics</td><td>31850</td><td></td></tr><tr><td>0.2 &amp;micro;m pore-size Acrodisc syringe filter</td><td>VWR</td><td>4192</td><td></td></tr><tr><td>10 mL Luer-lok syringe</td><td>VWR</td><td>BD309604</td><td></td></tr><tr><td>60 mL Luer-lok syringe</td><td>VWR</td><td>BD309653</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>FischerScientific</td><td>S233-500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>ThermoScientific</td><td>C79-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>EMD Millipore</td><td>5980</td><td></td></tr><tr><td>HBSS w/o Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;, w/o phenol red, Gibco</td><td>ThermoScientific</td><td>14175-095</td><td></td></tr><tr><td>MEM w/o phenol red, Gibco</td><td>ThermoScientific</td><td>5100-038</td><td></td></tr><tr><td>Horse serum, heat-inactivated</td><td>ThermoScientific</td><td>26050-070</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>VWR</td><td>16777-164</td><td></td></tr><tr><td>Ultrapure low melting point agarose</td><td>ThermoScientific</td><td>16520-100</td><td></td></tr><tr><td>ethanol, absolute</td><td>ThermoScientific</td><td>BP2818-4</td><td></td></tr><tr><td>2-phenoxyethanol</td><td>Sigma</td><td>77699</td><td></td></tr><tr><td>Corning Cell-Tak cell and tissue adhesive&amp;nbsp;</td><td>VWR</td><td>354240</td><td></td></tr><tr><td>refractive index liquid&amp;nbsp;</td><td>Cargille Lab</td><td>1803Y</td><td></td></tr><tr><td>Nikon A1 multiphoton microscope equipped with a MaiTai infrared laser</td><td>Nikon</td><td></td><td>equivalent system will work</td></tr><tr><td>40X Apo long-distance water immersion objective (N.A. 1.15)</td><td></td><td></td><td></td></tr><tr><td>environmental chamber equipped with insert for 35 mm petridishes</td><td>Okolab</td><td></td><td>equivalent system will work</td></tr><tr><td>NIS analysis software</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

culture of adult transgenic zebrafish retinal explants for live-cell imaging by multiphoton microscopy

内源性再生程序是由穆勒神经胶质细胞在成年斑马鱼的神经元以下的损伤和死亡发起（斑马鱼 ）视网膜。的米勒胶质细胞重新进入细胞周期并产生经过随后的几轮细胞分裂并分化成丢失的神经元细胞类型的神经元祖细胞。既米勒胶质细胞和神经元祖细胞的细胞核复制它们的DNA，并且在描述为Interkinetic过程视网膜， 即它们的基内核层（INL）和外核层（ONL）之间分别迁移，的不同的位置进行有丝分裂核迁移（INM）。 INM已主要研究了在显影视网膜。审查INM在成人再生详细斑马鱼视网膜的动力学，需要荧光标记的米勒胶质细胞/神经元祖细胞的活细胞成像。在这里，我们提供给隔离条件和文化背视网膜从Tg为GFAP:nGFP]认为暴露于恒定的强光35 H mi2004斑马鱼。我们还表明，这些视网膜文化是可行的执行活细胞成像实验，利用多光子显微镜来监测GFAP的迁移行为在整个视网膜外植体的厚度是连续获得Z堆栈图像长达8小时:nGFP阳性细胞。此外，我们描述的细节来执行后成像分析，以确定根尖和基底INM的速度。总而言之，我们制定的条件来研究INM的动力学在神经再生的成年模型。这将推动我们的这一关键细胞过程的了解，使我们能够确定控制INM的机制。

根据图1中的示意性概述的过程的视网膜的隔离允许扁平背侧视网膜培养从光受损成人的Tg [GFAP:nGFP]在5％CO历时至少24小时的mi2004斑马鱼2 /空气环境。这些平面安装视网膜植可用于在深部组织的层次图像焦平面。一个例子是米勒胶质/标记与来自穆勒特定胶质启动子GFAP（胶质纤维酸性蛋白）的GFP神经元祖细胞的细胞核被在光损坏视网膜有丝分裂期间位于ONL（ 图2A - D，3）。 nt19斑马鱼眼睛下视杆细胞表达EGFP:为了进一步证实，这种方法适用于图像的各种视网膜细胞层，从完好的Tg为Eco.NfsB-EGFP RHO]准备急性分离的视网膜植视紫红质启动子。 图2E - H显示，有可能获得GFP阳性视杆和它们的内段的图像。作为棒内段朝视网膜色素上皮锥光感受器之间延伸，这些数据将意味着它也有可能图像锥光感受器。这是不是然而进一步调查。 具有观察米勒胶质细胞/神经元祖细胞中的ONL在光损坏视网膜细胞核，我们详细表征（ 图3和电影1）他们的迁移行为。米勒胶质细胞/神经元祖细胞由绿色荧光蛋白标记的细胞核迁移双向的INL，他们通常驻留和ONL，感光体损的部位的基底位置之间。通常情况下，GFAP:nGFP阳性细胞核移植的INL短距离（ 图3A - C）他们经历了前核膜破裂，同时仍然位于INL，基于绿色荧光蛋白的再分配到整个米勒胶质细胞/神经元祖细胞（ 图3D）的细胞质中。继外核层细胞分裂，二GFAP:nGFP阳性核成为在回到基础INL（ 图3H - J）外核层（ 图3F，G）可见。当细胞分裂时，新产生的核起初非常暗淡，因此难以辨认（ 图3G）。然而，有丝分裂的一到两个时间框架内，核GFP荧光变得明亮了，这使核移植的可视化（ 图3H - J）。活细胞成像也允许的分割面（ 图3F，G），该水平发生视网膜用于图3中所示的细胞的顶端表面的可视化。它也是可行使用其它转基因斑马鱼线，例如作为Tg [GFAP:EGFP]表达在细胞质中的GFP NT11斑马鱼（未示出数据）。虽然可以可靠地确定心尖运动和水平的细胞分裂，它往往很难准确识别基部迁移子细胞核和垂直细胞分裂中的T g的位置[GFAP:EGFP] NT11斑马鱼视网膜（数据未显示）。 为了确定速度，一个GFAP:nGFP阳性细胞核未在整个录音期间迁移被选为参考点（明星， 图4A - E）。 -在最大XZ或图像系列的每个时间点nGFP阳性细胞核（E水平红线， 图4A）:在相对于参考GFAP测定-所述转移核（E垂直红线， 图4A）的距离YZ-突起（取决于在哪些角度晶核可以visuali捷思最有效）。该核迁移的距离被暗算的电子表格（ 图4F -高 ）的时间。核膜破裂常常在图中作为一个初始basalward运动观察由于在整个小区内的GFP的再分布（在此实例中的单元主体的基部大部分）。心尖速度为核膜破裂前确定，拟合线性回归曲线（灰色虚线， 图4G）来暗算的时候了核膜破裂前的时间段（红钻， 图4G）的距离。线性回归曲线（表达式y = mx + c）的斜率的"m"表示速度"DX / dt的&#39;。对于在图4中呈现的细胞的顶端速度为10.89微米/小时。同样的方法用于计算初始快速基底运动速度的新形成的子细胞核的（V B）（ 图4F， </str翁&gt; H）。的子细胞D1和D2的-67.71和33.51微米/小时基底迁移速度V B，分别为同等比根尖速度更快。不幸的是，这是不可能的，以确定核膜破裂作为整个细胞扩散的GFP后根尖速度。取而代之的是，瞬时速度是基于核膜破裂和新形成的子细胞核的第一可视化前核的定时和位置来计算。的7.9微米/小时用于图4中所示的单元中的瞬时速度是可能是低估的染色质到达ONL之前末期有丝分裂的，当子细胞核可见。 总结，视网膜外植体的培养可以在成人再生斑马鱼视网膜和迁移和细胞分裂PA的判定INM的活细胞成像个体细胞核rameters。 <img alt="图1" src="/files/ftp_upload/55335/55335fig1.jpg" /> 图1:视网膜分离过程。 一 ）示意图说明斑马鱼的头与它的眼睛。 B）的眼睛从斑马鱼取出并导向显示眼睛与瞳孔的前面。 C）转动眼球180°，使眼与视柄（填充黑色圆圈）后面的（D变为可见）和瞳孔朝下。灰度颜色表示眼睛的巩膜的存在。 E）的一对麦弗逊式Vannas剪刀的一个刀片被插入眼睛的视柄（实心黑色圆圈）将其切割成其背侧和腹侧边这是由白色分割线表示。 F）眼背一半去除腹后半段。黑色半圈为T他对视柄的残留部分。 G）的转动眼90°向后以显示（H）的眼睛的内部与仍然由巩膜（灰线包围的视网膜（棕色），玻璃体（未示出）和透镜（L，浅灰色圆圈） ）。 我 ），巩膜被分离（注意，灰线缺失）和J）的晶状体和玻璃体去掉引起仅视网膜（棕色）。 K）视网膜旋转90°向前引起背视网膜（L）的平面式安装视图。 M）背视网膜外植体平板安装在玻璃底FluoroDish中。 <a href="http://ecsource.jove.com/files/ftp_upload/55335/55335fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55335/55335fig2.jpg" /> 图2:3-D重建视网膜多光子Z-堆栈。 A）多光子Z堆栈图像序列GFAP的三维重建:nGFP-阳性细胞的斑马鱼视网膜全安装的文化光损伤后48 h。 乙- D）GFAP单多光子XY-图片:在ONL（B）中，INL（C），相当于包含穆勒层的水平在（A三维重建显示）nGFP阳性细胞神经胶质细胞胞体和保利协鑫（D）。箭头（B）表示扩大一轮米勒的神经胶质细胞在ONL核膜破裂后。 E）多光子Z堆栈图像序列从完好的Tg视网膜外植体的三维重建[RHO:Eco.NfsB-EGFP] nt19斑马鱼。 的F -高 ）单多光子XY-图像在杆内段（RIS，F）中 ，杆的核层的水平（G），并在视网膜内层（H）的电平。 GCL，神经节细胞层; INL，内核层; ONL，外核层; RIS，杆内节。比例尺在A，D，E和H，10微米。 <a href="http://ecsource.jove.com/files/ftp_upload/55335/55335fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55335/55335fig3.jpg" /> 图3:INM的延时摄影图片系列。 A - j）图片系列Z堆栈时光倒流系列三维重建显示Tg为GFAP:nGFP] mi2004的经历INM中（外核层视网膜外植体上排）来划分阳性细胞核。 1顶部迁移核和其基部迁移子细胞核的位置由红色箭头表示。红星有丝分裂过程表明核膜破裂。 一个- J，底部行），相同的图像系列作为最上面一行是过饱和和裁剪集中在ONL分裂细胞。 INL，内核层; IPL，内网层; ONL，外核层。比例尺在A，10微米，是B中的相同- J. <a href="http://ecsource.jove.com/files/ftp_upload/55335/55335fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55335/55335fig4.jpg" /> 图4:顶回和底偏移速度分析。 A - E）的z栈图像序列的Tg的最大YZ投影[GFAP:nGFP]在由多光子显微镜获得的缩时拍摄的不同时间点mi2004视网膜外植体。使用"人工测量&#39;作用下的行工具，一个水平线被放置在基准小区的水平，由星号表示。垂直我asurement线被定位起始于水平线并延伸到迁移GFAP的基部位置:nGFP阳性核。测量线的长度在白盒上的图像和用于图像下可读性中给出。 L1的子细胞迁移1 =长度; L2的子细胞迁移2 =长度。 F）距离该小区（F0）在（A测量- E）顶部迁移，而且所产生的子细胞D1和D2在多光子慢速拍摄记录基部迁移。红线指示将其计算出的心尖迁移速度（V a）当黑色和灰色线表示用于计算基础迁移速度为D1和D2（V B），分别测量的测量结果。 G，H）的距离被暗算了核膜破裂（G之前心尖迁移在Excel中的时间）和快速的基础的阶段出于对女儿的单元格D1（H）的迁移。线性回归曲线拟合和功能都与代表速度的斜率的曲线图下方给出。 INL，内核层; IPL，内网状层; ONL，外核层。 <a href="http://ecsource.jove.com/files/ftp_upload/55335/55335fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="2"> TG [GFAP:EGFP] NT11 </td><td colspan="2"> Tg为RHO:Eco.NfsB-EGFP] nt19 </td></tr><tr><td></td><td> 激光功率 </td><td> 获得 </td><td> 激光功率 </td><td> 获得 </td></tr><tr><td>顶（视杆） </td><td> 13 </td><td> 126 </td><td> 3.5 </td><td> 118 </td></tR&gt; <tr><td>中东（穆勒神经胶质细胞） </td><td> 10 </td><td> 126 </td><td> 2.8 </td><td> 118 </td></tr><tr><td>底部（GCL） </td><td> 8 </td><td> 126 </td><td> 2.1 </td><td> 118 </td></tr></tbody></table> 表1:激光增益电平用于Z-强度修正在不同的飞机为图2显示的实验- 4。 <img alt="图2" src="/files/ftp_upload/55335/55335mov1.jpg" /> 电影1:由多光子显微镜视网膜外植体活细胞成像。 <a href="https://www.jove.com/files/ftp_upload/55335/Lahne_et_al_Supplemental_movie_JOVE.avi">（点击下载）。</a>

Watch this Scientific Journal Video about Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy at JoVE.com

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

斑马鱼视网膜再生大多被使用固定的视网膜研究。然而，再生反应过程中发生的动态过程，如interkinetic核迁移，并需要活细胞成像研究的基本机制。这里，我们描述培养和成像条件下，使用多光子显微术来监控Interkinetic核迁移（INM）的实时性。

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

culture-adult-transgenic-zebrafish-retinal-explants-for-live-cell

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.5K 次观看.  University of Notre Dame. 斑马鱼视网膜再生大多被使用固定的视网膜研究。然而，再生反应过程中发生的动态过程，如interkinetic核迁移，并需要活细胞成像研究的基本机制。这里，我们描述培养和成像条件下，使用多光子显微术来监控Interkinetic核迁移（INM）的实时性。 

视频: Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

科研

发育生物学

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

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 ...

生物学

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

神经科学

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

斑马鱼光学杯形态形成的四维成像

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

神经元发育过程中的 ROS 活细胞成像

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...