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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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+cytoplasmic+cofilin-actin+rods.">Isolation and characterization of cytoplasmic cofilin-actin rods.</a> Journal of Biological Chemistry. 285 (8), 5450-5460 (2010).</li><li>Masurovsky, E. B., Benitez, H. H., Kim, S. U., Murray, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin,+development,+and+nature+of+intranuclear+rodlets+and+associated+bodies+in+chicken+sympathetic+neurons.">Origin, development, and nature of intranuclear rodlets and associated bodies in chicken sympathetic neurons.</a> The Journal of Cell Biology. 44 (7), 172-191 (1970).</li><li>Feldman, M. L., Peters, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranuclear+rods+and+sheets+in+rat+cochlear+nucleus.">Intranuclear rods and sheets in rat cochlear nucleus.</a> Journal of Neurocytology. 1 (2), 109-127 (1972).</li><li>Nishida, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+is+a+component+of+intranuclear+and+cytoplasmic+actin+rods+induced+in+cultured+cells.">Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells.</a> Cell Biology. 84 (8), 5262-5266 (1987).</li><li>Ono, S., Abe, H., Nagaoka, R., Obinata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+ADF+and+cofilin+in+intranuclear+actin+rods+of+cultured+muscle+cells.">Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells.</a> Journal of Muscle Research and Cell Motility. 14 (2), 195-204 (1993).</li><li>Xue, Z., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back-to-back+mechanisms+drive+actomyosin+ring+closure+during+Drosophila+embryo+cleavage.">Back-to-back mechanisms drive actomyosin ring closure during Drosophila embryo cleavage.</a> The Journal of Cell Biology. 215 (3), 335-344 (2016).</li><li>Cao, J., Albertson, R., Riggs, B., Field, C. M., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuf,+a+Rab11+effector,+maintains+cytokinetic+furrow+integrity+by+promoting+local+actin+polymerization.">Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization.</a> Journal of Cell Biology. 182 (2), 301-313 (2008).</li><li>Spracklen, A. J., Fagan, T. N., Lovander, K. E., Tootle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pros+and+cons+of+common+actin+labeling+tools+for+visualizing+actin+dynamics+during+Drosophila+oogenesis.">The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.</a> Developmental Biology. 393 (2), 209-226 (2014).</li><li>Chen, Q., Nag, S., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formins+filter+modified+actin+subunits+during+processive+elongation.">Formins filter modified actin subunits during processive elongation.</a> Journal of Structural Biology. 177 (1), 32-39 (2012).</li><li>Iordanou, E., Chandran, R. R., Blackstone, N., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+interference+by+dsRNA+injection+into+Drosophila+embryos.">RNAi interference by dsRNA injection into Drosophila embryos.</a> Journal of Visualized Experiments. (50), 2477 (2011).</li><li>Juarez, M. T., Patterson, R. A., Li, W., McGinnis, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+wound+assay+and+in+vivo+localization+of+epidermal+wound+response+reporters+in+Drosophila+embryos.">Microinjection wound assay and in vivo localization of epidermal wound response reporters in Drosophila embryos.</a> Journal of Visualized Experiments. 81, 50750 (2013).</li><li>Carreira-Rosario, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombineering+homologous+recombination+constructs+in+Drosophila.">Recombineering homologous recombination constructs in Drosophila.</a> Journal of Visualized Experiments. (77), 50346 (2013).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+techniques+for+studying+mitosis+in+the+Drosophila+melanogaster+syncytial+embryo.">Microinjection techniques for studying mitosis in the Drosophila melanogaster syncytial embryo.</a> Journal of Visualized Experiments. (31), 1382 (2009).</li><li>Catrina, I. E., Bayer, L. V., Omar, O. S., Bratu, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+and+tracking+endogenous+mRNAs+in+live+Drosophila+melanogaster+egg+chambers.">Visualizing and tracking endogenous mRNAs in live Drosophila melanogaster egg chambers.</a> Journal of Visualized Experiments. (148), 58545 (2019).</li><li>Wessel, A. D., Gumalla, M., Grosshans, J., Schmidt, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+properties+of+early+Drosophila+embryos+measured+by+high-speed+video+microrheology.">The mechanical properties of early Drosophila embryos measured by high-speed video microrheology.</a> Biophysical Journal. 108 (8), 1899-1907 (2015).</li><li>Mollinari, C., Gonz&#225;lez, A., Cid-Arregui, A., Garc&#237;a-Carranc&#225;, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+and+transgenesis.">Microinjection and transgenesis.</a> Microinjection and Transgenesis: Strategies and Protocols. , 587-603 (1998).</li><li>Figard, L., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+shape+change+in+living+Drosophila+embryos.">Imaging cell shape change in living Drosophila embryos.</a> Journal of Visualized Experiments. (49), 2503 (2011).</li><li>Oesterle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pipette Cookbook 2018: P-97 and P-1000 Micropipette Pullers. , (2018).</li><li>Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photographic+study+of+development+in+the+living+embryo+of+Drosophila+melanogaster.">A photographic study of development in the living embryo of Drosophila melanogaster.</a> Journal of Embryology and Experimental Morphology. 33 (3), 789-801 (1975).</li><li>Powsner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+temperature+on+the+durations+of+the+developmental+stages+of+Drosophila+melanogaster.">The effects of temperature on the durations of the developmental stages of Drosophila melanogaster.</a> Physiological Zoology. 8 (4), 474-520 (1935).</li><li>Hunter, M. 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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 border="0" cellpadding="0" cellspacing="0" style="width:837px;" width="838">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Adenosine triphosphate (ATP)</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">A23835G</td>
			<td style="width:299px;">Component of G buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Apple juice, Mott&#39;s, 64 fl oz</td>
			<td style="width:195px;">Mott&#39;s</td>
			<td style="width:121px;">014800000344</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bacto Agar</td>
			<td style="width:195px;">BD</td>
			<td style="width:121px;">214010</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Bleach, PureBright Germicidal, 6.0% sodium hypochlorite</td>
			<td style="width:195px;">KIK International</td>
			<td style="width:121px;">059647210020</td>
			<td>For dechorionating embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Calcium chloride</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">C1016500G</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell strainer, 70 &#956;m</td>
			<td style="width:195px;">Falcon</td>
			<td style="width:121px;">352350</td>
			<td>For collecting dechorionated embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Confocal microscope, LSM 880 34-channel with Airyscan</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">0000001994956</td>
			<td>For imaging intranuclear actin rods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Desiccant</td>
			<td style="width:195px;">Drierite</td>
			<td style="width:121px;">24001</td>
			<td>For desiccating embryos</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Dissecting microscope, Stemi 508 Stereoscope with 8:1 zoom</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4350649000000</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dissecting needle, 5 in</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">08965A</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dithiothreitol (DTT)</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">BP1725</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Double-sided Tape, Scotch Permanent, 0.5 in x 250 in</td>
			<td style="width:195px;">3M</td>
			<td>021200010323</td>
			<td>For making embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Embryo collection cage</td>
			<td style="width:195px;">Genessee Scientific</td>
			<td style="width:121px;">59100</td>
			<td>For housing adult flies and collecting embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Fine tip tweezers, Dumont Tweezer, Style 5</td>
			<td style="width:195px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">72701D</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Glass capillaries, Borosillicate glass, thin 1 mm x 0.75 mm</td>
			<td style="width:195px;">World Precision Instruments, Inc.</td>
			<td style="width:121px;">TW1004</td>
			<td>For microneedles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Halocarbon oil 27</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">H8773100ML</td>
			<td>For hydration of embryos</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:223px;">Heated stage incubator</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4118579020000, 4118609020000, 4118609010000</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:223px;">Lab Tissue Wipers, KimWipes</td>
			<td style="width:195px;">Kimberly-Clark</td>
			<td style="width:121px;">34155</td>
			<td>Lab tissue wipers</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Light microscope, Invertoskop 40C Inverted Phase contrast microscope, refurbished</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">Discontinued</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Methyl-4-hydroxybenzoate</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">H36471KG</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microinjector, FemtoJet4x</td>
			<td style="width:195px;">Eppendorf</td>
			<td>5253000025</td>
			<td>Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Micro loader tips, epT.I.P.S. 20 &#956;L</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:121px;">5242956003</td>
			<td>For loading microneedles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Micromanipulator and injection stage with x,y,z dials for needle adjustment</td>
			<td style="width:195px;">Bernard Instruments, Inc (Houston, TX)</td>
			<td style="width:121px;">Custom</td>
			<td>For performing microinjections</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Micropipette puller, Model P-97, Flaming/Brown</td>
			<td style="width:195px;">Sutter Instruments</td>
			<td style="width:121px;">P97</td>
			<td>For pulling capillary tubes to make microneedles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Microscope cover glass 24x50-1.5</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">12544E</td>
			<td>For mounting embryos</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Microscope slides, Lilac Colorfrost, Precleaned, 25 x 75 x 1mm</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">22037081</td>
			<td>For mounting embryos for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">n-Heptane</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">H3601</td>
			<td>Component of embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Objective, 10x</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">Discontinued</td>
			<td>10x objective for injection microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Objective, C-Apochromat 40x/1,2 W Korr. FCS</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4217679971711</td>
			<td>40x water objective for confocal</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">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 style="width:195px;">Zeiss</td>
			<td style="width:121px;">4208529871000</td>
			<td>25x mixed immersion objective for confocal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Objective, Plan-Apocrhomat 63x/1.40 Oil DIC f/ELYRA</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4207829900799</td>
			<td>63x oil objective for confocal</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Paintbrush, Robert Simmons Expression E85 Pointed Round size 2</td>
			<td style="width:195px;">Daler-Rowney</td>
			<td style="width:121px;">038372016954</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Paper towels, Kleenex C-fold paper towels, white</td>
			<td style="width:195px;">Kimberly-Clark</td>
			<td style="width:121px;">884266344845</td>
			<td>For blotting cell strainer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pasteur pipette, 5 3/4 in</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">1367820A</td>
			<td>For covering embryos with oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Petri dish, glass, 100 x 20 mm</td>
			<td style="width:195px;">Corning</td>
			<td style="width:121px;">3160102</td>
			<td>For humid incubation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Petri dish, plastic, 60 x 15 mm</td>
			<td style="width:195px;">VWR</td>
			<td style="width:121px;">25384092</td>
			<td>For apple juice plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Pipette, Eppendorf Reference 0.5-10 &#956;L</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:121px;">2231000604</td>
			<td>For loading the microneedle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pipette tip, xTIP4 250 &#956;L</td>
			<td style="width:195px;">Biotix</td>
			<td style="width:121px;">63300006</td>
			<td>For adding embryo glue to coverslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Razor blade</td>
			<td style="width:195px;">VWR</td>
			<td style="width:121px;">55411050</td>
			<td>For cutting agar wedge, tape, pipette tips</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Rhodamine-conjugated globular actin, human platelet (non-muscle; 4x10 &#956;g)</td>
			<td style="width:195px;">Cytoskeleton, Inc.</td>
			<td style="width:121px;">APHR-A</td>
			<td>G-actin^Red</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Scintillation vial, 20 mL Glass borosillicate with polyethylene liner and urea caps</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">033377</td>
			<td>For making embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Screw top jar, 16 oz</td>
			<td style="width:195px;">Nalgene</td>
			<td style="width:121px;">000194414195</td>
			<td>For desiccating embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Stage micrometer</td>
			<td style="width:195px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">602104PG</td>
			<td>For calibrating volume of G-actin injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Sucrose</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">840971KG</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trizma base</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">T15031KG</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Yeast, Lesaffre Yeast Corporation Yeast, Red Star Active Dry, 32 oz</td>
			<td style="width:195px;">Lesaffre Yeast Corporation</td>
			<td style="width:121px;">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

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

斑马鱼胚胎的倍性操作与热休克治疗2

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. Here, we describe in detail our current protocol for the dissection of Drosophila larval brains, and then present our current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for high-resolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. We also present custom-made macros that we routinely use to correct for drifting and to extract and process specific quantitative information from time-lapse analysis.

Applications of Immobilization of Drosophila Tissues with Fibrin Clots for Live Imaging

This protocol describes applications of sample immobilization using Fibrin clots, limiting drifting, and allowing the addition and washout of reagents during live imaging. Samples are transferred to a drop of Fibrinogen containing culture medium on the surface of a coverslip, after which polymerization is induced by adding thrombin.

使用纤维素凝块固定 的果蝇 组织用于实时成像

Drosophila is an important model system to study a vast range of biological questions. Various organs and tissues from different developmental stages of ...

成像核内作用棒在活热应力 果蝇 胚胎

成像核内作用棒在活热应力果蝇胚胎