Name: imaging intranuclear actin rods in live heat stressed drosophila embryos
Uploaded: 2020-05-15T14:30:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos at JoVE.com

作者感谢郑柳柳和薛增辉（在索卡克实验室帮助开创这项技术）以及帮助分析的哈桑·西德（Hasan Seede）的工作。这项研究的工作由国家卫生研究院（R01 GM115111）的赠款资助。

1. 准备胚胎收集杯和苹果汁琼脂盘
<ol><li>在注射实验前五天，建造28 个或采购至少两个小胚胎收集杯。制作新鲜的60毫米苹果汁琼脂板，用于小收集杯28。在4°C时用湿纸巾覆盖的塑料盒中的存储板。 注:小胚胎收集杯，填充了第1.3步描述的苍蝇数量，将提供足够的胚胎数量每个实验，同时也确保胚胎处理和注射可以在足够短的时间内完成，使早期发育阶段的成像。</li>
	<li>将苹果汁盘加热至18°C，并在盘子中央加入一点酵母膏。酵母糊是活性酵母和蒸馏水的简单糊。</li>
	<li>为了促进最慷慨的产卵，在实验前2天用苍蝇设置收集杯。将至少100只雌性苍蝇和50只雄性苍蝇添加到收集杯中，并配上准备好的苹果汁盘（图1，第1步）。在注射实验之前的几天里，每天至少换两次苹果汁盘，一次在早上，一次在晚上。 注:当胚胎收集杯保持在18°C，并具有12小时的开灯/光关闭周期时，将获得最佳的注射和成像结果。</li>
</ol>2. 为微弹射准备 G-actin红色 工作库存解决方案
注:此制剂仅生产 G-actinRed5 mg/mL 工作库存的 2 μL，因此，如果用户不习惯微击技术，则可以跳过第 3 步，使用中性 pH 缓冲器练习微注射，以保存宝贵的工作库存。供应商的 10 微克 G-actinRed 库存可存放在 16 盎司螺丝顶罐中，在 4 °C 下用 500 克干燥剂存放长达 6 个月。
<ol><li>提前准备一个 G 缓冲库存解决方案: 5 mm 特里斯 - Hcl， 0.2 mm Cacl2，ph 8.0 。在室温下过滤和存储。</li>
	<li>在注射当天，使用第 2.1 步的 G 缓冲液库存在冰上使用新的捕捉帽微中心管中准备 1 mL 的 G 缓冲工作液，并在指示的最终浓度下补充以下:1 m 二硫三醇 （DTT） 和 0.2 mM ATP，pH 8.0。 注:G缓冲工作解决方案的1mL体积超过实验所需的量，但简化了准备。实验后可以丢弃多余的，或者用户可以根据自己的喜好缩小规模。<ol>
<li>将 10 μg G-actin红色 股票保留在冰上，首先在管内 G-actinRed 的粉红色液滴顶部添加 1 μL 过滤蒸馏水。</li>
		<li>接下来，从步骤 2.2 添加 1 μL 的冷、新鲜准备的 G 缓冲工作解决方案。向上和向下管道~20次，与设置为1μL的移液器音量很好地混合。G-actin红 股票现在将在最后稀释5毫克/mL。</li>
	</ol>
</li>
	<li>在不受干扰的冰上孵化准备好的G-actin红色 30分钟。</li>
	<li>将准备好的 G-actinRed 在微中心中以 16，000 x g 为 20 分钟、在 4 °C 下进行离心，以去除任何沉淀物。</li>
	<li>小心地将 1.5 μL 的超纳坦放入冰上新鲜的捕捉帽微中心管中，避免深粉色颗粒。</li>
	<li>将准备好的 G-actin红色 超强剂储存在冰上长达 6 小时，直到准备加载到微需要中。 注:微食水可以提前从毛细管中拉出微点拉拔器，然后在室温下储存在 100 x 20 mm Petri 盘中的建模粘土条上。建议的微尼拔参数可以在派珀特食谱29中找到。</li>
</ol>3. 收集胚胎并安装注射
<ol><li>允许苍蝇在苹果汁盘上放置胚胎30分钟，酵母糊在18°C。</li>
	<li>当苍蝇铺设时，预先加热苹果汁琼脂板 ，不将酵母糊粘贴 到室温下，用剃须刀刀片切出4厘米×1厘米的苹果汁琼脂矩形楔子，放在25毫米×75毫米的玻璃滑梯上。<ol>
<li>通过倒入新鲜漂白剂，用蒸馏水稀释1:1，在盘子上旋转1分钟，从而从30分钟的采集中收获盘子，并去除胚胎，如28（图1，第2步）所述。 注:不同品牌的漂白剂以不同浓度出售。这里使用的漂白剂是瓶中6%的次氯酸钠，被稀释为3%的次氯酸钠。其他浓度稍低的漂白剂品牌同样有效。</li>
		<li>将漂白剂和被分离的胚胎倒入收集篮（70μm细胞过滤器），用喷出的瓶子中的蒸馏水冲洗两次，将这些洗涤液加入收集篮中。</li>
		<li>用蒸馏水大力冲洗收集篮中残缺的胚胎，直到看不到酵母团，篮子在纸巾上弄脏时不会留下多余的漂白剂的粉红色痕迹。</li>
	</ol></li>
	<li>使用用蒸馏水浸湿的刷毛的画笔，将收集篮中的浸出和洗净的胚胎转移到玻璃滑梯上准备好的苹果汁琼脂楔子上。</li>
	<li>使用一对细尖钳或解剖针，沿着长方形琼脂楔子的长轴（图1，步骤3）将十个胚胎排列成一条直线。将胚胎从头到尾排列，使胚胎前极朝右，正对着研究人员（图1，步骤3，放大）。</li>
	<li>用剃须刀刀片切断P200移液器尖端的0.5厘米，浸入"胚胎胶水"（28中描述）。慷慨地涂上一个区域 5 毫米宽 （图 1，步骤 4）沿 24 毫米 x 50 毫米矩形封面的长边， 让干燥， 胶水侧向上。干燥需要~30s，一旦整个胶涂层区域出现哑光而不是潮湿或有光泽，干燥就会完成。 注:提前至少48小时准备"胚胎胶水"。在闪烁的瓶中将n-Heptane添加到双面胶带条中，如28年所述。</li>
	<li>一旦"胚胎胶水"干涸，轻轻地将盖片胶面放在一排对齐的胚胎上，在盖片边缘和一排胚胎之间留下2-3毫米的空间。 注意:将胚胎粘在离盖片边缘太近的地方可能导致胚胎在实验过程中大量干燥。</li>
	<li>翻转盖片，使胚胎现在朝上。它们应该卡在沿着盖片的一条长边的一条线上，其腹腔区域面对着盖片的最近边缘（图1，第4步，放大）。</li>
	<li>将带胚胎的盖片轻轻地放在储存在16盎司螺丝顶部罐中的150克新鲜蓝色干燥剂上，使胚胎脱脂。紧紧拧在盖子上，孵育8-10分钟（图1，第5步）。</li>
	<li>干燥后，从干燥罐中取出盖片，将盖片的每个短侧胶带移到显微镜滑动，胚胎侧面向上，用两块4厘米2的双面胶带使胚胎盖片贴入注射阶段（图1，步骤6）。</li>
	<li>加入2-3滴卤碳27油与巴斯德移液器覆盖对齐的胚胎，并保护他们免受进一步脱水（图1，步骤6）。</li>
</ol>4. 注射和加热应激胚胎，促进作用杆形成
注:所有注射均在18°C的温度控制室进行。
<ol><li>准备湿润的孵化室从玻璃培养皿至少100毫米×20毫米的大小和线与扭曲的实验室组织雨刷器与蒸馏水（图1，步骤8）。在注射胚胎之前，在32°C或所需的孵化温度前预热孵化室。</li>
	<li>打开微型喷射器的气流阀，打开微型喷射器（压力至少为 90 psi 的压缩空气或内部空气是合适的）。</li>
	<li>当胚胎干燥时，使用微型装载机尖端将先前制备的G-actin红色 超强剂倒入微需要。设置移液器以绘制 1-1.5 μL。 注:由于行为素的粘度，加载量可能不准确，可能有足够的作用素来加载至少一到两个微需要。如果微食量校准正确，并且在实验过程中不堵塞，每个加载的微注射器最多可注射60个胚胎。</li>
	<li>将微针连接到针架上并拧紧螺钉。将气管连接到微喷射器，确保微呼吸平衡上的回流压力达到 30 hPa。</li>
	<li>校准微注射器设置，在幻灯片微米上排出直径为 100μm 的 G-actinRed 气泡 （+500 pL）。在微容器上旋转压力旋钮（500-1500 hPa）和注脉冲时间旋钮（0.1-0.5 s），以获得正确的气泡大小。每次加载新的微内窥镜时，都会调整这些设置，以考虑行为粘度和微细端尺寸的变异性。 注:制备的G-actin红 是粘稠的，在注射胚胎之前，微针尖端可能有空气应被排出。如果 G-actinRed 不容易从微尼德的尖端排出，则轻轻将微线尖端从滑动微米的边缘打破。</li>
	<li>将装有胚胎的幻灯片放在显微镜阶段。 注:每次注射设置将有所不同，因此研究人员将不得不相应地调整他们的注射方法。在这里，胚胎被移动到固定的微针，通过运行胚胎到微针注射每个胚胎。</li>
	<li>调整微操纵器阶段，将10倍目标的焦点放在光显微镜上，使胚胎可见。当葡萄线膜的轮廓最锋利，胚胎出现最大时，胚胎处于正确的焦点平面中。选择处于正确发育阶段的胚胎注射，以便在注射后孵化完成时，大部分离合器达到所需的发育阶段（例如，在Bownes的阶段2-330 注射胚胎，以便观察在32°C的热应激后细胞化棒）。</li>
	<li>使用微针控制，将针头带入与胚胎相同的聚焦平面。 注意:如果微针在移动舞台或微针时在盖片上捕获，则微针离滑动太近，不在正确的焦距平面中。微针应与盖夹平行，而不是以显著的角度（图1，步骤7）。</li>
	<li>将微需要插入胚胎中，使其在胚胎"赤道"处击中其腹腔区域中间的胚胎。当微型针尖在胚胎中间可见时，用脚踏板或"注射"按钮触发注射（图1，步骤7）。</li>
	<li>注射一次G-actin红色 ，慢慢去除微针。移动舞台，为适当的发育阶段的每个胚胎重复。 注:胚胎的膨胀是正常的，因为注射了G-actin红 ，一点细胞质可能会从胚胎中泄漏出来。</li>
	<li>所有胚胎注射完后，将带胚胎的滑梯放在制备的潮湿孵化室中，并合上盖子（图1，步骤8）。如果试图获得达到细胞化的胚胎，热应力在32°C的胚胎在潮湿的孵化室60-75分钟。 注:潜伏时间被注意到，允许在细胞化热应力胚胎的棒可视化。非热应激控制胚胎的潜伏时间会更长，因为在较低的温度31下发育会变慢。这些控制胚胎可以在潮湿的室中孵育，温度为18°C或25°C，具体实验的设计和要问的问题。G-actin红 在胚胎中扩散所需的最短潜伏时间为30分钟。</li>
</ol>5. 通过分泌显微镜在高温受压胚胎中的图像作用棒
<ol><li>当胚胎受到热压时，打开包膜显微镜，选择561nm激光通道。</li>
	<li>将目标透镜（建议为 25 倍、40 倍或 63 倍）移到工作位置。</li>
	<li>如果成像热应力胚胎，则设置加热阶段孵化器，以达到32°C的内部温度。 点对点或红外温度计可用于检查目标处或附近的温度。</li>
	<li>孵化完成后，用注射的胚胎从潮湿的孵化室中取出滑梯（图1，步骤9）。</li>
	<li>工作迅速，轻轻地撬开双面胶带片，用于粘附盖片与安装胚胎到幻灯片（图1，步骤9）。 注意:在这些步骤中要温和，因为如果施加过多的力，盖片很容易破碎。</li>
	<li>将两块2.5厘米长的双面胶带粘在一起，将胶带切成两半，制作两条，长2.5×0.5厘米（图1，步骤10）。</li>
	<li>将每个胶带长度的三分之二贴在第一个盖片上，在 Halocarbon 27 油中胚胎的两侧（图 1，步骤 10，橙色），留下三分之一的胶带条挂在胚胎卡住的第一个盖片的边缘。在这一步中，使用戴手套的手，小心不要触摸胚胎。</li>
	<li>轻轻地将第二个矩形盖片放在胶带条上，将胚胎夹在盖片之间（图1，步骤10，蓝色）。对齐 25 毫米边缘，但保持 50 毫米边缘相互偏移 1 厘米宽度。 注:这第二个盖片的全表面将成为新的成像表面，将面对客观的镜头，所以要小心不要得到指纹或卤素27油在这第二个盖片表面。偏移是必要的，以便额外的卤素27油可以添加到均匀浸入胚胎。如有必要，在三明治顶部两个盖片相遇的接缝处加入 Halocarbon 27 油，它会通过毛细管作用覆盖胚胎（见图 1中的虚线，第 10 步）。</li>
	<li>轻轻轻点直接在胶带顶部的盖片区域，用剃须刀的钝侧让盖片粘附在胶带上。</li>
	<li>翻转盖滑三明治，放在实验室组织雨刷器上，以保持成像表面清洁，并携带到结肠显微镜（图1，第11步）。在成像前，确保磁带完全粘在两个盖片上。</li>
	<li>确认加热阶段处于温度，如果使用倒置显微镜，请在选定的客观透镜上添加浸入液。</li>
	<li>小心地将盖片三明治放在舞台上，以确保新的成像表面（2和盖片）是触摸浸入式液体（图1，步骤11）。 注意:如果需要，请将盖片三明治用两小块双面胶带粘在舞台上，以防止在成像过程中出现不必要的移动，如果盖片不适合加热阶段。</li>
	<li>聚焦于处于细胞化（Bownes阶段4a30）或使用传播光或荧光进行发育阶段的胚胎。</li>
	<li>胚胎聚焦后，切换到显微镜上的激光采集模式，并根据需要调整激光功率和增益、帧大小、平铺和投影设置。</li>
	<li>通过胚胎核的焦点平面拍摄表面图像，以找到核内部作用杆。棒应以多个方向显示为相对较暗的原子核（图 2A，2C）内的亮条纹或点。</li>
</ol>6. 替代成像实验
<ol><li>执行 FRAP，以调查沿核作用杆长度或细胞质作用素结构（如细胞化过程中等离子膜皱眉的尖端）的变换。</li>
	<li>在成像软件中，选择一个围绕沟尖或行动棒的矩形区域来获取实验。</li>
	<li>选择矩形采集区域内行为杆中心的小方形区域进行漂白。确保作用杆的尖端是可见的，在实验过程中不要漂白，以便准确跟踪细胞核内的杆。</li>
	<li>将漂白激光器设置为 50 倍，并将漂白激光功率设置为最大。</li>
	<li>选择每秒获取图像的时间过程，以最高像素的停留速度总共达到 120 秒，并在图像采集的前两秒后将激光设置为漂白。</li>
	<li>量化数据，以确定荧光恢复的一半时间1。</li>
</ol>

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没有宣布利益冲突。

这种方法的意义在于，它利用了在果蝇胚胎21、22、23、24、25、26、27中建立的微射方案，使有关ASR和伴随作用杆组装的新研究得以进行。将G-actinRed注射到活胚胎中的一个主要优点是，ASR可以在各种背景下进?...

该协议描述了如何注射G-actinRed，以可视化在热应力胚胎中核作用杆的组装，这些胚胎正在经历不可诱导的Actin应激反应（ASR）1。我们开发了这个协议，以帮助研究ASR，在胚胎导致破坏形态生成和降低生存能力，并在成人细胞类型与病理学，包括肾衰竭2，肌肉肌病3，阿尔茨海默氏症和亨廷顿舞蹈症4，5，6，7，8。这种ASR是由许多细胞应力引起的，包括热休克9，10，11，氧化应激4，6，减少ATP合成12，和异常亨廷丁或β淀粉样寡头化4，5，6，7，9，13，14，15，16。ASR的一个标志是在受影响细胞的细胞质或细胞核中组装异常作用素棒，这是由压力引起的活性相互作用蛋白，Cofilin1，5，6，10的过度激活驱动的。不幸的是，在ASR方面仍然存在关键的知识差距。例如，行为棒的功能尚不清楚。我们不明白为什么棒在某些细胞类型的细胞质中形成，但其他细胞核。也不清楚ASR是保护性的还是对承受压力的细胞或胚胎的不适应。最后，我们仍然不知道科菲林多活化或作用杆组装背后的详细机制。因此，该方案提供了一个快速和多才多艺的检测，通过可视化活果蝇胚胎高度可处理的实验系统中的活杆形成和动力学来探测ASR。
将G-actinRed微导入活体果蝇胚胎的方案最初是为了研究组织构建事件期间正常细胞质作用素结构17的动态而开发的。在这些研究中，我们发现G-actin红注射不会对胚胎的早期发育过程产生负面影响，包括细胞因子或胃痛17，18。然后，我们修改了协议，调整了胚胎处理和G-actin红注射，以允许在进行ASR1的热应激胚胎中成像作用素棒。除G-actin红注射外，其他方法可用于在胚胎中可视化作用素。这些方法依赖于表达荧光蛋白（FPs）标记为作用素或作用素结合蛋白的领域，如营养素-mCherry、生命-生命-GFP和莫辛-GFP（在19年审查）。然而，使用这些FP探头需要谨慎，因为它们可以稳定或破坏一些行为结构，不等于标记所有行为结构20，在actin-GFP的情况下，是高度过度表达-有问题的杆组装分析，这不仅是压力依赖，而且行动浓度依赖1。因此，G-actinRed是苍蝇胚胎中棒研究的首选探针，胚胎的大尺寸允许其轻松注射。
该协议的工作流程类似于其他成熟的微注射技术，这些技术已被用于将蛋白质、核酸、药物和荧光指标注射到果蝇胚胎21、22、23、24、25、26、27。然而，在G-actinRed的微注射之后，胚胎暴露在轻度热应激下，诱导ASR和核内作用杆组装。对于能够接触苍蝇和注射钻机的实验室，这种方法应易于实施，并适应与ASR有关的具体研究路线，包括它通过不同的应力或在不同遗传背景的调制而诱导。

<table><tbody><tr><td>Adenosine triphosphate (ATP)</td><td>Millipore-Sigma</td><td>A23835G</td><td>Component of G buffer</td></tr><tr><td>Apple juice, Mott&#039;s, 64 fl oz</td><td>Mott&#039;s</td><td>014800000344</td><td>Component of apple juice plates</td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td>Component of apple juice plates</td></tr><tr><td>Bleach, PureBright Germicidal, 6.0% sodium hypochlorite</td><td>KIK International</td><td>059647210020</td><td>For dechorionating embryos</td></tr><tr><td>Calcium chloride</td><td>Millipore-Sigma</td><td>C1016500G</td><td>Component of G buffer</td></tr><tr><td>Cell strainer, 70 &amp;mu;m</td><td>Falcon</td><td>352350</td><td>For collecting dechorionated embryos</td></tr><tr><td>Confocal microscope, LSM 880 34-channel with Airyscan</td><td>Zeiss</td><td>0000001994956</td><td>For imaging intranuclear actin rods</td></tr><tr><td>Desiccant</td><td>Drierite</td><td>24001</td><td>For desiccating embryos</td></tr><tr><td>Dissecting microscope, Stemi 508 Stereoscope with 8:1 zoom</td><td>Zeiss</td><td>4350649000000</td><td>For arranging embryos on agar wedge</td></tr><tr><td>Dissecting needle, 5 in</td><td>Fisher Scientific</td><td>08965A</td><td>For arranging embryos on agar wedge</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Fisher Scientific</td><td>BP1725</td><td>Component of G buffer</td></tr><tr><td>Double-sided Tape, Scotch Permanent, 0.5 in x 250 in</td><td>3M</td><td>021200010323</td><td>For making embryo glue</td></tr><tr><td>Embryo collection cage</td><td>Genessee Scientific</td><td>59100</td><td>For housing adult flies and collecting embryos</td></tr><tr><td>Fine tip tweezers, Dumont Tweezer, Style 5</td><td>Electron Microscopy Sciences</td><td>72701D</td><td>For arranging embryos on agar wedge</td></tr><tr><td>Glass capillaries, Borosillicate glass, thin 1 mm x 0.75 mm</td><td>World Precision Instruments, Inc.</td><td>TW1004</td><td>For microneedles</td></tr><tr><td>Halocarbon oil 27</td><td>Millipore-Sigma</td><td>H8773100ML</td><td>For hydration of embryos</td></tr><tr><td>Heated stage incubator</td><td>Zeiss</td><td>4118579020000, 4118609020000, 4118609010000</td><td>For confocal imaging</td></tr><tr><td>Lab Tissue Wipers, KimWipes</td><td>Kimberly-Clark</td><td>34155</td><td>Lab tissue wipers</td></tr><tr><td>Light microscope, Invertoskop 40C Inverted Phase contrast microscope, refurbished</td><td>Zeiss</td><td>Discontinued</td><td>Injection microscope</td></tr><tr><td>Methyl-4-hydroxybenzoate</td><td>Millipore-Sigma</td><td>H36471KG</td><td>Component of apple juice plates</td></tr><tr><td>Microinjector, FemtoJet4x</td><td>Eppendorf</td><td>5253000025</td><td>Microinjector</td></tr><tr><td>Micro loader tips, epT.I.P.S. 20 &amp;mu;L</td><td>Eppendorf</td><td>5242956003</td><td>For loading microneedles</td></tr><tr><td>Micromanipulator and injection stage with x,y,z dials for needle adjustment</td><td>Bernard Instruments, Inc (Houston, TX)</td><td>Custom</td><td>For performing microinjections</td></tr><tr><td>Micropipette puller, Model P-97, Flaming/Brown</td><td>Sutter Instruments</td><td>P97</td><td>For pulling capillary tubes to make microneedles</td></tr><tr><td>Microscope cover glass 24x50-1.5</td><td>Fisher Scientific</td><td>12544E</td><td>For mounting embryos</td></tr><tr><td>Microscope slides, Lilac Colorfrost, Precleaned, 25 x 75 x 1mm</td><td>Fisher Scientific</td><td>22037081</td><td>For mounting embryos for injection</td></tr><tr><td>n-Heptane</td><td>Fisher Scientific</td><td>H3601</td><td>Component of embryo glue</td></tr><tr><td>Objective, 10x</td><td>Zeiss</td><td>Discontinued</td><td>10x objective for injection microscope</td></tr><tr><td>Objective, C-Apochromat 40x/1,2 W Korr. FCS</td><td>Zeiss</td><td>4217679971711</td><td>40x water objective for confocal</td></tr><tr><td>Objective, LD LCI Plan-Apochromat 25x/0.8 Imm Cor DIC M27 for oil, water, silicone oil or glycerine immersion (D=0-0.17mm) (WD=0.57mm at D=0.17mm)</td><td>Zeiss</td><td>4208529871000</td><td>25x mixed immersion objective for confocal</td></tr><tr><td>Objective, Plan-Apocrhomat 63x/1.40 Oil DIC f/ELYRA</td><td>Zeiss</td><td>4207829900799</td><td>63x oil objective for confocal</td></tr><tr><td>Paintbrush, Robert Simmons Expression E85 Pointed Round size 2</td><td>Daler-Rowney</td><td>038372016954</td><td>For transferring embryos</td></tr><tr><td>Paper towels, Kleenex C-fold paper towels, white</td><td>Kimberly-Clark</td><td>884266344845</td><td>For blotting cell strainer</td></tr><tr><td>Pasteur pipette, 5 3/4 in</td><td>Fisher Scientific</td><td>1367820A</td><td>For covering embryos with oil</td></tr><tr><td>Petri dish, glass, 100 x 20 mm</td><td>Corning</td><td>3160102</td><td>For humid incubation chamber</td></tr><tr><td>Petri dish, plastic, 60 x 15 mm</td><td>VWR</td><td>25384092</td><td>For apple juice plates</td></tr><tr><td>Pipette, Eppendorf Reference 0.5-10 &amp;mu;L</td><td>Eppendorf</td><td>2231000604</td><td>For loading the microneedle</td></tr><tr><td>Pipette tip, xTIP4 250 &amp;mu;L</td><td>Biotix</td><td>63300006</td><td>For adding embryo glue to coverslip</td></tr><tr><td>Razor blade</td><td>VWR</td><td>55411050</td><td>For cutting agar wedge, tape, pipette tips</td></tr><tr><td>Rhodamine-conjugated globular actin, human platelet (non-muscle; 4x10 &amp;mu;g)</td><td>Cytoskeleton, Inc.</td><td>APHR-A</td><td>G-actin^Red</td></tr><tr><td>Scintillation vial, 20 mL Glass borosillicate with polyethylene liner and urea caps</td><td>Fisher Scientific</td><td>033377</td><td>For making embryo glue</td></tr><tr><td>Screw top jar, 16 oz</td><td>Nalgene</td><td>000194414195</td><td>For desiccating embryos</td></tr><tr><td>Stage micrometer</td><td>Electron Microscopy Sciences</td><td>602104PG</td><td>For calibrating volume of G-actin injection</td></tr><tr><td>Sucrose</td><td>Millipore-Sigma</td><td>840971KG</td><td>Component of apple juice plates</td></tr><tr><td>Trizma base</td><td>Millipore-Sigma</td><td>T15031KG</td><td>Component of G buffer</td></tr><tr><td>Yeast, Lesaffre Yeast Corporation Yeast, Red Star Active Dry, 32 oz</td><td>Lesaffre Yeast Corporation</td><td>117929157002</td><td>Component of yeast paste</td></tr></tbody></table>

imaging intranuclear actin rods in live heat stressed drosophila embryos

该协议的目的是可视化在热应激后在活 的果蝇黑色素酯 胚胎中组装的核内作用棒。Actin 棒是伴随人类病理（包括神经退行性疾病）的保存、诱导的阿克廷应激反应 （ASR） 的标志。以前，我们表明ASR会导致形态发生失败，降低胚胎的存活率。该协议允许在高度适应成像、遗传学和生物化学的模型系统中继续研究作用杆组装和ASR的基础机制。胚胎被收集并安装在盖片上，为注射做准备。罗达明结合球蛋白（G-actin红）被稀释并加载到微需要中。每个胚胎的中心只注射一次。注射后，胚胎在高温下孵育，然后通过分泌显微镜对核细胞内作用杆进行可视化。光漂白（FRAP）实验后的荧光恢复可以在作用杆上进行:细胞质中其他富含作用的结构也可以成像。我们发现，G-actin红 聚合物像内源性G-actin，本身并不干扰正常的胚胎发育。此协议的一个限制是，在注射过程中必须小心谨慎，以避免对胚胎造成严重伤害。然而，在实践中，将G-actinRed 注射到 果蝇 胚胎中是一种快速可靠的可视化作用杆的方法，可以很容易地用于任何基因型的苍蝇或引入其他细胞应力，包括缺氧和氧化应激。

图1中描绘了胚胎处理的示意图工作流程，并在表1中介绍了典型实验的时间表。一个好的实验结果的估计是，每注射10个胚胎，至少一半的被观察的胚胎将处于正确的发育阶段，没有损坏，并表现出强大的ASR与热应力在32°C。 图2A（右面板）中胚胎的代表性表面视图图像中所示，将通过核内作用素棒的组装来证明此 ASR。Actin 棒将出现在原子?...

Watch this Scientific Journal Video about Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos at JoVE.com

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

该协议的目标是将罗达明结合球蛋白注射到 果蝇 胚胎中，并在热应力后将图像核作用杆组装。

成像核内作用棒在活热应力 果蝇 胚胎

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

imaging-intranuclear-actin-rods-live-heat-stressed-drosophila

Research

JoVE Journal

Developmental Biology

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

3.4K 次观看.  Baylor College of Medicine. 该协议的目标是将罗达明结合球蛋白注射到 果蝇 胚胎中，并在热应力后将图像核作用杆组装。

视频: Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

直立成像<em&gt;果蝇</em&gt;蛋钱伯斯

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

科研

发育生物学

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

在活动区域​​的解剖和影像果蝇神经肌肉交界处

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

神经科学

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

在成像钙<em&gt;果蝇</em&gt;在蛋激活

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

生物学

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

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

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

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

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

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

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

精子发生的细胞学分析：Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

成像核内作用棒在活热应力果蝇胚胎

摘要

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直立成像

在活动区域的解剖和影像果蝇神经肌肉交界处

在成像钙

在体内果蝇蛹肌肌腱形态的影像学研究

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

在完整的果蝇幼虫体内的亚细胞分辨率成像

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

GFP标记的蛋白质在实时成像

精子发生的细胞学分析：Live和固定制剂

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

成像核内作用棒在活热应力 果蝇 胚胎

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此视频中的章节

直立成像

在活动区域​​的解剖和影像果蝇神经肌肉交界处

在成像钙

在体内果蝇蛹肌肌腱形态的影像学研究

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

在完整的果蝇幼虫体内的亚细胞分辨率成像

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

GFP标记的蛋白质在实时成像

精子发生的细胞学分析：Live和固定制剂

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

成像核内作用棒在活热应力果蝇胚胎

在活动区域的解剖和影像果蝇神经肌肉交界处

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络