共焦显微镜是在美国国家科学基金会 (NSF) 的资助下购买的, 赠款 #DBI-0722569。这项研究得到了美国国立卫生研究院/国家综合医学研究所 (NIH/研究院) 赠款 #GM085290 和美国国防部 (DOD) 赠款 #W81XWH-16-1-0466 授予 rm 布鲁斯特。重要的是通过前学院和本科生科学教育计划, 授予 #52008090 UMBC 从霍华德休斯医学院的赠款支持。某地布朗得到美国教育部 GAANN 奖学金的支持, 约瑟夫·迈尔霍夫研究生奖学金由 NIH/研究院补助金 #GM055036 资助, 以及由美国国防部拨款 #W81XWH-16-1-0466 资助的研究助理。

道德声明: 下面描述的程序遵循马里兰大学巴尔的摩县动物保育指南. 
 1. 使用 Immunolabeling 的稳定和动态 MTs 的分析 (协议 1) <ol> <li> 手动 dechorionation 前固定的胚胎 ( <ol> <li> 通过浇注多余的系统水来获得新鲜的胚胎)然后将剩余的胚胎收集到塑料培养皿中 (参见 材料表 ). </li>
		<li> 清除系统水中的任何碎片, 将胚胎移植到一个充满了胚胎培养基的新皿中 (参阅 材料表 ), 以确保胚胎在清洁的环境中发育. </li>
		<li> 允许胚胎在28.5 和 #176 温度控制的恒温箱中发育到所需的阶段; C. </li>
		<li> 在 dechorionation 之前, 将小于24小时胚乳 (hpf) 的胚胎放置在玻璃盘中. 
		注: Dechorionate 前的胚胎固定, 以最大限度地快速渗透的固色剂和保存 MT 完整性。使用胚胎培养基代替系统水, 在 dechorionation 期间提供所需的额外 Ca 2 + . </li>
		<li> 在培养皿中, 用细钳在解剖显微镜下手动取出胚胎中的 chorions。
		<ol> <li> 在圆形的透明绒毛膜中捏出一个小区域, 用一对镊子将胚胎围住, 并将镊子轻轻地拉开, 在膜上形成破裂. </li>
			<li> 用镊子巧妙地窥探破裂的绒毛膜, 扩大开口。注意不要用钳子碰触胚胎, 因为它可能破裂. </li>
		</ol> </li> </ol> </li> <li> 分阶段胚胎的固定, dechorionated 胚胎移植到1.5 毫升离心管。使用玻璃巴斯德吸管移除尽可能多的胚胎培养基. 
		注: 对年轻 (mid-somitogenesis) 胚胎进行固定和药物治疗, 在形成神经中心调解疼痛感觉之前, 不需要额外程序来减轻安乐死期间的疼痛。发展阶段的定义是在 et al ., 1995 25 。4-5 和11-12 节阶段用于获取 图 2 和 3 的图像。
		<li> 准备4% 甲醛 (粉煤灰)/MT 组装缓冲 (单克隆人) 固定剂 (参见 材料表), 将1毫升8% 的每1毫升2X 单克隆抗体组合在一起, 并添加2和 #181; 每100% 毫升 X-100 总体积. 
		注意: 在处理含有粉煤灰和海卫 X-100 的溶液时戴上手套, 这是皮肤刺激物. </li>
		<li> 将1毫升4% 的胚胎固定在28.5 和 #176 的5分钟; c. 用吸管吸出定影液, 用1毫升新鲜定影液代替, 在摇杆上室温 (RT) 孵育 3 h. 
		注: 样品必须在其生物温度 (28.5 #176; 斑马鱼 C) 中快速固定, 以防止温度依赖性 MT 降解. </li>
	</ol> </li> <li> 吸定影液并添加1毫升1x 的三缓冲盐水与 NP40 (TBS-NP40) 缓冲器。轻轻地在摇杆上搅动三次, 每次5分钟。将胚胎储存在4和 #176; C 在1毫升新鲜 1X TBS-NP40, 不超过7天. 
	注意: 在处理含有 NP-40 的溶液时戴上手套, 皮肤刺激性. </li>
	<li> 剖切胚胎为 immunolabeling <ol> <li> 热 RT 4% 低熔点 (LMP) 琼脂糖嵌入介质在一个封闭的容器, 直到解决方案变得清晰使用热板设置为50和 #176; C 定位接近解剖显微镜.保持容器在样品之间闭合并且在整个嵌入过程中加热 (步骤 1.4. 4-1. 4.6). </li>
		<li> 使用玻璃吸管将胚胎从1.5 毫升离心管转移到培养皿中, 然后用 1X TBS-NP40 填充. </li>
		<li> 在解剖显微镜的放大倍数下, 用细钳从 somitogenesis 阶段胚胎 (4-5 和11-12 节) 中取出大的卵黄细胞 26 。用一副钳子将胚用尾芽夹住, 并将蛋黄细胞与另一对一起剥开, 以保存后脑组织。将 de-yolked 胚胎转移到培养皿中无蛋黄碎片的区域. 
		注: 在琼脂糖填充模中嵌入胚胎, 以防止 LMP 琼脂糖的过早硬化. </li>
		<li> 填充一个 12 mm x 5 mm x 3 mm 的剖切模具与200和 #181; 我用微融化了 LMP 琼脂糖执行步骤 1.4. 5. 1.4. 6。迅速 (在二十年代内填充模具) 在 LMP 琼脂糖冷却到 RT 和固化前植入胚胎. </li>
		<li> 用细钳将 tailbud 从培养皿中的 de-yolked 胚转移到琼脂糖填充的模具, 并在解剖显微镜下将其锥形端. </li>
		<li> 使用细钳将胚胎定向到模具中, 使 vibratome 在所需的平面上切割。通过定向胚胎来创建横断面, 使后脑组织与模具的长度平行, 其背表面朝向边缘, 其前表面朝向锥形区域末端。对其余的胚胎重复步骤 1.4. 4-1. 4.6. </li>
		<li> 允许琼脂糖嵌入在 RT 中固化5分钟. </li>
		<li> 生成40和 #181; m 部分的最高轴琼脂糖嵌入胚胎 (步骤 1.4. 1-1. 4.7) 使用 vibratome 与切片菜填充 1x TBS-NP40。将感兴趣的部分转移到500和 #181 的24井板; L 1x TBS-NP40 使用细钳。每井只放置一个胚胎的部分. 
		注意: 有关详细信息, 请参阅参考 18 。确保在至少250和 #181 的情况下始终保持水分, 以低速 (10-25 rpm) 的缓冲和岩石的 L 为剩余的步骤, 以防止分离琼脂糖嵌入。在堵塞和洗涤液中存在的洗涤剂应减少液体介质的表面张力, 并允许浸入部分。检查在所有操作期间和之后, 部分留在井中。请小心避免在洗涤过程中意外丢弃节. </li>
	</ol> </li> <li> 删除缓冲区并添加500和 #181; L 阻塞解决方案。在 RT 上至少有1小时的岩石. 
	注: 使用包含每种二次抗体宿主种类的5% 血清的阻断溶液 (参见 材料表 ). </li>
	<li> 在300和 #181 中孵育; 在4和 #176 中, 主要抗体在阻塞缓冲液中稀释36-72 小时; C 在摇杆上。在600和 #181 洗两次; L 1x TBS-NP40 在一个摇杆上, 每次30分钟, 在 RT. 
	注: 对总 mts (反和 #946;-蛋白, 或反和 #945;-蛋白) 和稳定 mts (反谷氨酸-蛋白) 或动态 mts (反 Tyr 蛋白) 的主抗体孵化的双标签部分。选择主要的抗体, 已提出在不同的寄主物种时, 双标记的总和 post-translationally 修饰和 #945; 蛋白的人口。有关抗体稀释, 请参阅 材料表 . </li>
	<li> 在300和 #181 中孵育; 荧光共轭二级抗体在16-24 小时的摇杆上稀释, 在4和 #176; C 在黑暗中。在600和 #181 洗两次; L 1x TBS-NP40 在一个摇杆上, 每次30分钟, 在 RT. 
	注: 从这一点开始, 在每次操作后, 用铝箔包裹含有二次抗体的井碟, 以防淬火。选择与主抗体的宿主免疫球蛋白反应的二级抗体.选择具有独立、不重叠发射光谱的二次抗体荧光。有关抗体稀释, 请参阅 材料表 . </li>
	<li> 孵育500和 #181 中的胚胎; DAPI 溶液在一个摇杆上30分钟, 在 rt 上 TBS-NP40 三次, 在 rt 中为5分钟的晃动. 
	注: 核标记为步骤1.12 中执行的 MT 量化提供了蜂窝上下文. </li>
	<li> 在无尘滑轨的中心放置一滴安装介质和防剂。使用优良的镊子将部分转移到安装介质滴。在样品顶部放置无尘片。将幻灯片存放在干燥、黑暗和凉爽的地方, 用铝箔包裹, 直到进行成像. 
	注: 在使用显微镜之前, 在幻灯片背面使用一个细尖的永久性标记来旋转部分, 有助于识别剖面. </li>
	<li> 共焦成像 <ol> <li> 在反向激光扫描共聚焦显微镜上安装剖面, 将幻灯片粘贴到舞台上, 片面向目标。确定控制幻灯片上适当的光学 (目标、激光和通道设置, 如增益和偏移), 并使它们在示例 27 之间保持一致。避免 oversaturating 像素以防止数据丢失. </li>
		<li> 使用选定的第二抗体荧光的通道设置捕获 Z 叠共聚焦图像, 并保存图像文件 27 。为每个部分获取 Z 堆栈. 
		注意: 使用以下获取设置复制用于获取 图 2 和 3 中的图像的参数: 模式 = XYZ;物镜放大倍数 = 63X 油浸透镜;目标数值孔径 = 1.4;Z 步 = 0.1 和 #181; m;Z 深度 = 16.23 和 #181; m. 使用以下通道设置: DAPI 励磁与20% 紫外线范围激光, 发射过滤器范围 = 430-480 nm, 光电倍增管 (pmt) 增益 = 525 V, 和 pmt 偏移 =-1.72%;448 nm 荧光 (参考 材料表 ) 励磁与 20% 488 nm 激光器, 发射过滤范围 = 493-573 nm, pmt 增益 = 689 V, pmt 偏移 =-0.2%;594毫微米荧光励磁与 32% 594 毫微米 laser, 发射过滤器范围 = 608-706 毫微米, pmt 增益 = 768 V 和 pmt 偏移量 =-6.8%. </li>
		<li> 使用唯一的描述性文件名保存原始数据文件, 并在图像分析软件中创建用于编辑的副本. </li>
	</ol> </li> <li> 编译 Z 堆栈以显示最大投影 <ol> <li> 使用公共域3维图像分析软件 ( 如 、ImageJ) 打开数据文件副本。检查每个通道是否显示为单个图像序列 (Z 堆栈). </li>
		<li> 使用以下菜单序列分割图像通道: 和 #8220; 图像/颜色/分割通道和 #8221;. </li>
		<li> 通过使用以下菜单序列覆盖感兴趣的通道来创建合并图像: #34; 图像/颜色/合并通道. #34; 选择 594 nm、488 nm 和 DAPI 通道分别为红色、绿色和蓝色。检查和 #34; 创建复合 #34; 选择和 #34; 确定 #34; 28 . 
		注意: 省略 DAPI 通道以在 图 2 和 3 的最大投影中更好地传递特定于 MTs 的详细信息, 方法是仅为其他两个通道选择假颜色. </li>
		<li> 检查合并的 z 堆栈, 并记下所有可见通道的内部最佳 z 平面的开始和结束位置。关闭通常具有次优信号的外部 Z 平面, 这是由于剖面的不均匀表面造成的。有关详细信息, 请参阅参考 29 . </li>
		<li> 将合并的 z 堆栈可视化为单个2维图像, 方法是使用以下3维图像分析菜单序列执行 z 堆栈的最大强度投影: 和 #34; 图像/堆栈/Z 项目. #34; 输入内部最佳的起始和结束位置Z-平面从步1.11.3 作为和 #34; 开始切片和 #34; 和 #34; 停止切片, #34 分别。选择和 #34; 最大强度和 #34; 作为投影类型和点击和 #34; OK 和 #34;。有关详细信息, 请参阅参考 28 . </li>
	</ol> </li> <li> 分析 MT 标记 <ol> 打开商业3维图像分析软件。选择和 #34; 创建库和 #34; 并为图像库提供描述性名称。单击并 #34; 创建. #34; 将从共焦显微镜生成的原始图像文件拖动到库中。较大的文件需要更多的时间来传输. </li>
		<li> 选择要分析的文件。选择和 #34; 扩展焦点和 #34; 从和 #34; 查看和 #34; 菜单在主窗口中显示通道合并的图像. </li>
		<li> 通过向左或向右拖动每个通道的滑块工具来调整阈值, 直到降低背景信号并且真正的信号是可靠的。观察每个通道显示一个真实的信号的分子标记 ( 例如 , DAPI 通道显示长方形或有丝分裂核, 但不荧光从细胞质或琼脂糖). </li>
		<li> 选择 #34; 手绘感兴趣区域 (ROI) 和 #34; 工具和概述要分析的感兴趣的区域。选择和 #34; 操作和 #34; 选项卡, 后跟和 #34; 裁剪到选择和 #34; 裁剪图像。用新名称保存裁剪后的图像文件。单击 #34; 测量和 #34; 选项卡创建用于筛选与3维分析相关的特定对象的协议. </li>
		<li> 拖动和 #34; 查找对象和 #34; 到协议窗口。重命名第一个协议和 #34;D api. #34; 在下拉菜单中选择 DAPI 通道。将以下设置拖到 DAPI 协议中, 并将它们放在下面并 #34; 查找对象和 #34; 按以下顺序 ( 表 1 ): #34; 在对象和 #34 中填充孔;;#34; 单独的触摸物体和 #34;#34; 按大小和 #34 排除对象;;#34; 排除不接触 roi 和 #34;. 
		注意: 表 1 中设置的目标是首先设置一个阈值, 以丢弃其分布和大小与所分析对象的大小不一致的信号。例如, 在计算原子核时, 消除信号不太大, 不能成为原子核. </li>
		<li> 执行序列 (步骤 1.12.9) 将剩余的筛选器组合在一起 #946;-蛋白和其他标记使用 表 1 中的设置. </li>
		<li> 选择和 #34; 测量和 #34; 在每个协议的底部。选择和 #34; 强度和体积测量和 #34; 和 #34; 骨骼长度和 #34; 所有蛋白标签, 但只 DAPI 信号的前者. </li>
		<li> 在要测量的区域周围绘制一个 ROI。观察 #34 下的测量; 摘要和 #34; 软件处理区域后的选项卡。复制数据并将其保存到可行的电子表格中, 如 表 2 中所示。为以后的分析创建电子表格的备份副本. </li>
		<li> 选取感兴趣的测量值 (例如, mt 丛的长度、mt 丛的数目和核, 以显示不同的标记) 在电子表格和分析, 以确定每个组的平均值. 
		注: 平均 MT 丛长度 = 与 #39 的总和; #946 的平均值; 蛋白和 #39; 每个胚胎除以胚胎总数。请参阅 表 2 的20行。格式化电子表格, 使变量和实验组易于绘制. </li>
	</ol> </li> </ol> 2。 De 从头 MT 汇编分析 (协议 2) <ol> <li> 在实验前2天构造和测试井流设备 ( 图 1 ). 
	注: 该仪器可在使用来自 材料表 的 nocodazole 处理后同时冲洗多个实验组。硅胶封口机需要至少24小时的干燥时间, 才不会对胚胎产生毒性风险。
	<ol> <li> 使用夹具或带锯将50毫升离心管分成半纵长. </li>
		<li> 切割7半圆, 半径为3厘米, 出70和 #181; m 尼龙网布和修剪他们, 以适应紧密的一半分裂离心管。胶水的半圆到离心管平行于10毫升级标记使用水族馆安全的硅封口机。允许设备干燥2天, 并通过浸泡在一个烧杯的水2-3 小时冲洗. </li>
		<li> 将切割离心管的顶部 (螺纹) 端与建模粘土相线, 使流经该流体装置的液体的高度具有深度和 #188; 英寸 ( 图 1 , 视图2和 3). </li>
		<li> 通过从50毫升注射器中取出柱塞并将12英寸的细管插入尖端, 准备冲洗装置。推动油管, 只要它会去和密封周围的连接使用建模粘土. </li>
		<li> 预湿网格使用胚胎培养基, 使液体贯穿整个流动装置。将装置放在冰面上, 使所有车厢内的液体池都能倒出, 而在粘土轮辋所在的前部仍可清空。使用环形支架, 将冲洗装置挂在冰上流经的设备上方 ( 图 1 ). </li>
		<li> 冷却200毫升的胚胎培养基在冰上, 倒入足够的冲洗装置, 以确保所有的气泡被清除, 流速约7毫升/分钟. 通过改变注射器的高度来调节流速. </li>
	</ol> </li> <li> 酶 dechorionate 胚胎 <ol> <li> 在20毫升的胚胎培养基中稀释1毫升的10毫克/毫升非特异性蛋白酶, 从而制作出非特异蛋白酶的工作溶液. </li>
		<li> 在 timepoint 前1小时对胚胎进行化学 dechorionation, 期望它们达到预期的发育阶段。chorions 从100毫米培养皿中去除胚培养基, 加入20毫升的非特异性蛋白酶工作溶液. </li>
		<li> 在37和 #176 孵育胚胎; C 为 5 min. 
		注: 不超过5分钟或使用更高浓度的非特异性蛋白酶溶液, 因为这将导致的胚胎分裂一旦治疗与 nocodazole. </li>
		<li> 用大约25毫升的胚胎培养基快速移出非特异性蛋白酶和填充皿。重复一次. </li>
		<li> 使用1毫升玻璃吸管, 将小于 24 hpf 的胚胎移植到玻璃器皿中, 以保护它们免受损坏. </li>
		<li> 完成 dechorionation 通过手动删除 chorions 使用一对优良的镊子, 如步骤1.1.5 中所述. </li>
		<li> 在28.5 和 #176 中放置含有 dechorionated 胚胎的玻璃培养皿; C 孵化器至少30分钟, 直到达到预期的发育阶段. </li>
	</ol> </li> <li> 解现有 MTs <ol> <li> 准备一个5和 #181 的工作解决方案; nocodazole 50 和 #181 的结合; 1 毫克/毫升 nocodazole 与10毫升冰冷胚培养基. 
		注意: 使用手套时, 处理 nocodazole, 皮肤刺激性. </li>
		<li> 用10毫升冷 nocodazole 工作液交换 nocodazole 治疗组的胚培养基。将培养皿放在冰上, 为发育阶段 (例如, 4-5 节胚胎的1小时) 提供适当的时间。将未经处理的控制胚胎留在冰上的培养皿中, 并在步骤2.3.4.1 中与冲洗样品一起固定. </li>
		<li> 用火抛光1毫升玻璃吸管将胚胎移植到流经的设备, 每个实验组使用单独的隔间。启动 nocodazole 冲洗, 将冰冷胚培养基倒入50毫升注射器的顶部. 
		注: 每个实验组至少使用30胚胎。实验组可由控制胚胎或多种吗或 RNA 注入胚胎组成。冲洗将需要总共约150毫升的胚胎培养基将增加每8-10 分钟的 nocodazole, 同时继续抑制 MT 生长与冰。将胚胎保持在冰上对这种方法的成功至关重要, 因为 MTs 在低温下是不稳定的, 而且在早期胚胎中会出现冷的延迟. </li>
		<li> 允许 MTs 在 RT 中经过20分钟的冲洗后再生长, 将胚胎移植到含有温热 (28.5-#176; C) 的玻璃培养皿中, 使用火抛光1毫升玻璃吸管。胚胎一旦转移就开始计时
		<ol> <li> 将控制和冲洗胚胎在1分钟、5 min 和10分钟内, 由移大约10胚胎放入1.5 毫升的离心管中, 填充1毫升4% 的粉煤灰/单克隆 (28.5 和 #176; C), 并按照步骤1.2.3 中的指示进行操作. </li>
		</ol> </li> </ol> </li> <li> 为 immunolabeling 准备示例, 如1.3 至1.5 节中所述。
	<li> Immunolabel 漂浮部分和图像胚胎, 如1.6 节-1.10 所述, 主要抗体规格如下: 使用1:500 兔抗 #947;-蛋白和1:200 鼠抗 #946;-蛋白. </li>
	<li> 步骤1.12 中所述, 使用3维图像分析软件处理和分析图像. </li>
</ol>

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作者没有什么可透露的。

目前在斑马鱼的早期发展中有许多成像 MT 动力学的方法, 从标记分子的活体成像到固定组织的 immunolabeling11,12,13,14。虽然单个单元中的 mts 可以存在于动态或稳定状态, 但上皮是一个过程, 其中 mts 随着时间的推移而逐步稳定。使用稳定和动态 MTs 的标记提供了一种可视化这种现象的方法。这里提出的方?...

微管 (MTs) 是α和β-蛋白的聚合物, 组合成线性 protofilaments, 其中的几个组合成中空的管子1,2。MTs 是极化结构, 具有快速增长的加端和缓慢增长的负端, 锚定在中心或其他微管组织中心 (MTOC)3。De 从头mt 形成是由核在γ-蛋白环复合体 (γ-土耳其), 它提供一个模板为 mt 汇编4。在任何给定的单元格中, 两个 MTs 的种群可以区别开来, 以不同的速率翻转。动态 MTs 通过在称为动态不稳定的过程中切换增长阶段和收缩期来探索它们的蜂窝环境5。与动态 mts 不同, 稳定 mts 是 non-growing 的, 其半衰期比动态 mts6长。

几十年的细胞生物学研究提供了一系列复杂的工具来研究 MT 的结构和功能, 并在这些骨架元素上形成了大量的知识。例如, mts 在建立和维护细胞极性方面起着核心作用, 这不仅归因于它们的固有极性, 而且也归功于稳定与动态 mts 的微分亚空间分布7, 8. 相比之下, 在更复杂的三维 (3 维) 环境 (如脊椎动物胚胎) 中, 对 mt 体系结构和功能的了解要少得多, 部分原因是高分辨率下的 mt 细胞骨架成像的挑战。尽管这一限制, 最近一代的 GFP 表达的转基因线, 标签 mts 或瞬态表达的荧光标记 MT 标记已增加我们的理解的动态变化, mts 接受和他们的细胞和斑马鱼胚胎发育的作用。整个 MT 网络可以成像的转基因线, 其中蛋白是直接标记为9或蛋白聚合物是间接标记使用 MT 相关的蛋白 Doublecortin 样激酶 (Dclk) 或 Ensconsin (EMTB)10, 11。其他的线 (和构造) 已经产生, 通过专门标记 mt 的正负端或中心锚定的减号11,12,13,来评估 mt 的内在极性14. 这些工具的威力在于能够研究在活体和发育中的生物体的 MT 动力学。这些研究表明, 例如, 在特定细胞群体中 MTs 的空间和动态分布, 发生形态发生的组织中有丝分裂纺锤体的方向 (细胞分裂平面的指示器), MT 聚合物的极性因为它涉及到的过程, 如细胞伸长和迁移, 和 MT 生长率确定的彗星速度9,13,15。这些工具的局限性在于它们不容易区分稳定的和动态的 MT 种群。

本文从丰富的细胞生物学文献中, 对斑马鱼胚胎的图像稳定和动态 MTs 的 immunolabeling 方法进行了描述, 这是对转基因品系的补充。这种 immunolabeling 方法在斑马鱼中的广泛应用, 由于在固定过程中保持 MT 完整性的困难而受到了一定的阻碍。协议 1概述了在开发的斑马鱼后脑的横断面上 immunolabeling 总、动态和稳定 MTs 的优化方法。此外, 还描述了使用商用软件的直接方法来量化这些 MT 的数量。稳定 mts 的区别, 从动态 mts 的几个修饰修改的α-蛋白, 如乙酰化和 detyrosination, 在稳定的 mts 积累了一段时间16,17。在斑马鱼胚胎中, 乙酰化发生在睫状和轴突 mts 上, 但不在稳定的相间 mts18上, 将此标记的用处限制在稳定 mts 的子集上。相反, detyrosination 似乎发生在所有稳定的 MTs 在斑马鱼胚胎18。这种修饰修饰暴露了α-蛋白 (detyrosinated 蛋白)18的羧基末端谷氨酸, 并且可以使用反谷氨酸-蛋白19检测。虽然 detyrosination 优先于稳定 MTs, 但实验证据表明, 此修饰修改是由于 MT 稳定性16造成的, 而不是原因。相互 MT 人口, 由动态 MTs 组成, 是区分使用抗体, 反 Tyr-蛋白, 特别承认 tyrosinated 形式的α蛋白19。随着这些标记和共聚焦成像的 immunolabeling, 对 MTs (长度、数量和相对丰度) 的定量分析可以在发展中神经管的定义区域进行。这里提供了一种简化的方法, 用于使用3维图像处理软件执行此分析。此方法可用于解决有关形态发生和细胞极性的建立或成熟的问题20。的确, 稳定 MTs 的极化阵列的细化伴随着许多发展事件, 包括感光细胞发生形态21, 上皮在发育中的神经管的18和轴突形成8。

协议 2描述了一个用于在其组装阶段 (核化/锚定和生长)2223中分析 MTs 的细胞生物学检测的体内。初生的 MTs 在中心有核, 并随后锚定到母亲中心的 subdistal 附属物23。介绍了一种分析降解后初生 MT 再生的方法。本议定书详细介绍了解 MTs 的 nocodazole 治疗、药物冲洗程序和后处理恢复期。再生定期监测根据协议 1中所述的一般过程, immunolabeling 的后冲蚀与中心 (反γ-蛋白) 和原子核 (4 "、6-diamidino-2-吲 (DAPI)) 的标记一起与总 MTs (抗β-蛋白) 的标记。该协议的 mt 降解步骤是必不可少的, 因为它能够评估de 从头mt 的增长, 而不是预先存在的 MTs 的扩展。因此, 这一技术有别于其他公布的程序, 以测量 MT 生长速率 (在没有降解) 通过使用一个加号标记, 如最终结合蛋白3融合到绿色荧光蛋白 (EB3-GFP), 如在et al.中所示,201211。此外, 这种方法对于分析de 从头MT 组件中的缺陷尤其有用, 例如以前报告的NEDD1突变体, 在该变种中, 对中心的γ蛋白的征募被削弱, 导致不完整神经管形成和神经元缺陷24。

<table><tbody><tr><td>Agarose</td><td></td><td></td><td>Used to treat petridishes.&amp;nbsp;&amp;nbsp; Prepare 1% agarose&lt;br/&gt; by heating a solution of&amp;nbsp; 1 gram agarose per 100 ml 1X embryo medium in a microwave&lt;br/&gt; until polymerized.&amp;nbsp;</td></tr><tr><td>Kpipes</td><td>Sigma</td><td>P7643</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>T3253-500G</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333-500G</td><td></td></tr><tr><td>CaCl2&amp;middot;2H2O</td><td>Sigma</td><td>C5080</td><td></td></tr><tr><td>NP-40</td><td>American Bioanalyticals</td><td>AB01424</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E3889-25G</td><td></td></tr><tr><td>MgCl2</td><td>Sigma</td><td>M2670-500G</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher</td><td>BP1605</td><td></td></tr><tr><td>Triton-x</td><td>American Bioanalyticals</td><td>AB02025</td><td></td></tr><tr><td>Anti-Fade mounting medium</td><td>Invitrogen</td><td>P10144</td><td></td></tr><tr><td>Mouse anti-&amp;beta;-tubulin</td><td>Developmental studies Hybridoma Bank</td><td>E7</td><td>1/200</td></tr><tr><td>Rabbit anti-&amp;gamma;-tubulin</td><td>Genetex</td><td>GTX113286</td><td>1/500</td></tr><tr><td>Rabbit anti-&amp;alpha;-tubulin</td><td>Genetex</td><td>GTX108784</td><td>1/1000*</td></tr><tr><td>Rabbit anti-detyrosinated-tubulin</td><td>Millipore</td><td>AB3201</td><td>1/200-1/1000*&amp;nbsp; Titrate antibody with first use of new lot.</td></tr><tr><td>Rabbit anti-tyrosinated-tubulin</td><td>Millipore</td><td>ABT171</td><td>1/500</td></tr><tr><td>Mouse anti-centrin</td><td>Millipore</td><td>04-1624</td><td>1/1000</td></tr><tr><td>Goat 488 anti-rabbit</td><td>Thermofisher</td><td>A11008</td><td>1/500</td></tr><tr><td>Goat 594 anti-rabbit</td><td>Thermofisher</td><td>A11012</td><td>1/500</td></tr><tr><td>Goat 594 anti-mouse</td><td>Thermofisher</td><td>A11005</td><td>1/500</td></tr><tr><td>Goat 488 anti-mouse</td><td>Thermofisher</td><td>A11001</td><td>1/500</td></tr><tr><td>Vibratome</td><td>Vibratome</td><td>1500</td><td></td></tr><tr><td>Forceps</td><td>World Precision Instruments</td><td>555227F</td><td></td></tr><tr><td>100 mm petri dish</td><td>Cell treat</td><td>229693</td><td></td></tr><tr><td>35 mm petri dish</td><td>Cell treat</td><td>229638</td><td></td></tr><tr><td>50 ml falcon tube</td><td>Fisher</td><td>14-432-22</td><td></td></tr><tr><td>Woven nylon mesh 70 um</td><td>Amazon.com</td><td>B0043D1SZG&amp;nbsp;</td><td></td></tr><tr><td>Micropipette</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>Glass pipette</td><td>Fisher</td><td>NC-999363-9</td><td></td></tr><tr><td>Aquarium sealant</td><td>Amazon.com, by MarineLand</td><td>Silicone Sealer 1 oz (Tube)&amp;nbsp;</td><td></td></tr><tr><td>Ring stand</td><td>Fisher</td><td>14-675BO</td><td></td></tr><tr><td>Microbore PTFE Tubing, 0.022&quot;ID</td><td>Cole-Parmer</td><td>WU-06417-21</td><td></td></tr><tr><td>Modeling clay</td><td>Amazon.com</td><td>Sargent Art 22-4000</td><td>Any wax or oil based non-toxic modeling clay will suffice</td></tr><tr><td>Clamp</td><td>Fisher</td><td>02-215-466</td><td></td></tr><tr><td>60ml syringe</td><td>Fisher</td><td>14-820-11</td><td></td></tr><tr><td>Embryo medium (E3)</td><td></td><td></td><td>34.8 g NaCl&lt;br/&gt; 1.6 g KCl&lt;br/&gt; 5.8 g CaCl2&amp;middot;2H2O&lt;br/&gt; 9.78 g MgCl2&amp;middot;6H2O&lt;br/&gt; &lt;br/&gt; To prepare a 60X stock, dissolve the ingredients in H2O, to a final volume of 2 L. Adjust the pH to 7.2 with NaOH. Autoclave. To prepare 1X medium, dilute 16.5 mL of the 60X stock to 1 L.&amp;nbsp;</td></tr><tr><td>Blocking Solution</td><td></td><td></td><td>50 ml TBS-NP-40&lt;br/&gt; 2.5 ml normal goat serum&lt;br/&gt; 1 g BSA&lt;br/&gt; 625 &amp;micro;l Triton-X</td></tr><tr><td>TBS-NP-40 (pH 7.6)</td><td></td><td></td><td>155 mM NaCl&lt;br/&gt; 10 mM Tris HCl&lt;br/&gt; 0.1% NP-40</td></tr><tr><td>2x MAB (pH6.4)</td><td></td><td></td><td>160 mM KPIPES&lt;br/&gt; 10 mM EGTA&lt;br/&gt; 2 mM MgCl2</td></tr><tr><td>Commercial 3-D Image processing Software</td><td>PerkinElmer</td><td>Volocity (V 6.2)</td><td></td></tr><tr><td>Dry block heater&amp;nbsp;</td><td>VWR</td><td>12621-108</td><td>Used as a hot plate to melt agarose in Protocol 1.</td></tr><tr><td>Dissecting Microscope</td><td>Leica</td><td>MZ12</td><td></td></tr><tr><td>Confocal Microscope</td><td>Leica</td><td>SP5</td><td></td></tr><tr><td>Flat embedding mold</td><td>emsdiasum.com</td><td>BEEM 70904-01</td><td></td></tr><tr><td>Public domain image processing software</td><td>NIH</td><td>ImageJ (V 1.5)</td><td></td></tr><tr><td colspan="4">* Success varies by lot number</td></tr></tbody></table>

use of immunolabeling to analyze stable, dynamic, and nascent microtubules in the zebrafish embryo

微管 (MTs) 是动态的和脆弱的结构, 是挑战的图像在体内, 特别是在脊椎动物的胚胎。本文介绍了 Immunolabeling 方法, 分析了斑马鱼胚胎发育神经管中 MTs 的不同种群。虽然重点是神经组织, 这种方法是广泛适用于其他组织。程序为早期 mid-somitogenesis 阶段的胚胎 (1 节到12节) 进行了优化, 但是它们可以适应其他阶段的一系列相对较小的调整。第一个协议提供了一种评估稳定和动态 MTs 的空间分布的方法, 并用图像处理软件对这些种群进行定量分析。这种方法补充了现有的工具, 图像微管动力学和分布在 real-time, 使用转基因线或瞬态表达的标记结构。事实上, 这些工具非常有用, 但是它们并不容易区分动态和稳定的 MTs。图像和分析这些不同的微管种群的能力对于理解细胞极化和形态发生的机制有着重要的意义。第二个协议概述了专门分析初生 MTs 的技术。这是通过在一段时间内捕获 MTs 的de 从头生长特性来完成的, 这是随着药物 nocodazole 的微管降解和药物冲洗后的恢复期。这项技术尚未被应用于斑马鱼胚胎的 MTs 研究, 但它是一个有价值的方法, 以调查的体内功能的蛋白质牵连的微管组装。

基于 immunolabeling 的稳定动态 MTs 分析 在协议 1中, 利用谷氨酸-蛋白和 Tyr 蛋白作为稳定和动态 MTs 的标记, 揭示了神经管发育早期 (神经脊) 和晚期 (神经根) 阶段的 MT 亚的分布。动态 MTs 在神经龙骨阶段 (4-5 节) (图 2A-D) 的后脑中占主导地位。当龙骨发展成神经杆 (11-12 节), 增强上皮的阶段, 定性地较少 MTs 是免疫?...

Watch this Scientific Journal Video about Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo at JoVE.com

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Immunolabeling 的方法来分析不同群体的微管在发展斑马鱼大脑中描述, 这是广泛适用于其他组织。第一个协议概述了 immunolabeling 稳定和动态微管的优化方法。第二个协议提供了一种具体的图像和量化初生微管的方法。

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

use-immunolabeling-to-analyze-stable-dynamic-nascent-microtubules

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.2K 次观看.  University of Maryland, Baltimore County. Immunolabeling 的方法来分析不同群体的微管在发展斑马鱼大脑中描述, 这是广泛适用于其他组织。第一个协议概述了 immunolabeling 稳定和动态微管的优化方法。第二个协议提供了一种具体的图像和量化初生微管的方法。

视频: Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

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 ...

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

免疫染色磷酸化表位整装斑马鱼胚胎纤毛器官

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

化学

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

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

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

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

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

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

生物学

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

斑马鱼胚胎和幼虫的整个山免疫性化学

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

<meta charset="utf8"></head><body>斑马鱼胚胎的精细多视图光片显微镜实验方案

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

<meta charset="utf8"></head><body>作者聚焦:用于高分辨率多视图光片显微镜的斑马鱼胚胎安装策略 — 成像和图像重建技术

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

早期斑马鱼胚胎的光片显微镜成像和安装策略

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

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早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略