Name: use of immunolabeling to analyze stable, dynamic, and nascent microtubules in the zebrafish embryo
Uploaded: 2017-09-20T13:00:00
Description: Watch this Scientific Journal Video about Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo at JoVE.com

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...

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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。

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		<tr height="140">
			<td height="140" style="height:140px;width:207px;">Low Melting Point Agarose</td>
			<td style="width:177px;">IBI scientific</td>
			<td style="width:159px;">IB70050</td>
			<td style="width:373px;">Only used for embedding. 
			 
			Prepare 4% LMP agarose 
			by heating a solution of&#160; 4 grams LMP agarose per 100 ml 1X TBS in a microwave 
			until polymerized. Keep warm on a 50 &#730;C hot plate with the cap secured</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Agarose</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">Used to treat petridishes.&#160;&#160; Prepare 1% agarose 
			by heating a solution of&#160; 1 gram agarose per 100 ml 1X embryo medium in a microwave 
			until polymerized.&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Nocodazole</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">487928-10MG</td>
			<td style="width:373px;">Make stock by dissolving entire bottle of powder in distilled water and aliquoting into 500ul aliquots.&#160; Store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">8% Formaldehyde</td>
			<td style="width:177px;">Electron Microscopy Sciences</td>
			<td style="width:159px;">157-8-100</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Kpipes</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P7643</td>
			<td style="width:373px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:207px;">NaCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">S7653</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Tris-HCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">T3253-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">KCl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P9333-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">CaCl2&#183;2H2O</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">C5080</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">NP-40</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB01424</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">EGTA</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">E3889-25G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">MgCl2</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">M2670-500G</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Normal Goat Serum</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">S26-100ml</td>
			<td style="width:373px;">Aliquot and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Bovine serum albumin (BSA)</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">BP1605</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Triton-x</td>
			<td style="width:177px;">American Bioanalyticals</td>
			<td style="width:159px;">AB02025</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">Pronase E (non-specific Protease from Streptomyces griseus)</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:159px;">P5147-1G</td>
			<td style="width:373px;">make stock solution by diluting 10mg per ml of ddH2O. Aliquot in 1ml aliquots and store at -20 &#730;C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Anti-Fade mounting medium</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">P10144</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">DAPI</td>
			<td style="width:177px;">Invitrogen</td>
			<td style="width:159px;">D1306</td>
			<td style="width:373px;">Prepare 50 ml DAPI solution by first combining 1 &#181;l DAPI stock&#160; with 1 ml sterile water and then adding the DAPI/water mix to 49 ml 1X TBS; store in foil at 4 &#730;C for months.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:207px;">Mouse anti-&#946;-tubulin</td>
			<td style="width:177px;">Developmental studies Hybridoma Bank</td>
			<td style="width:159px;">E7</td>
			<td style="width:373px;">1/200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#947;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX113286</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Rabbit anti-&#945;-tubulin</td>
			<td style="width:177px;">Genetex</td>
			<td style="width:159px;">GTX108784</td>
			<td style="width:373px;">1/1000*</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-detyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">AB3201</td>
			<td style="width:373px;">1/200-1/1000*&#160; Titrate antibody with first use of new lot.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Rabbit anti-tyrosinated-tubulin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">ABT171</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Mouse anti-centrin</td>
			<td style="width:177px;">Millipore</td>
			<td style="width:159px;">04-1624</td>
			<td style="width:373px;">1/1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11008</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-rabbit</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11012</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 594 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11005</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Goat 488 anti-mouse</td>
			<td style="width:177px;">Thermofisher</td>
			<td style="width:159px;">A11001</td>
			<td style="width:373px;">1/500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Vibratome</td>
			<td style="width:177px;">Vibratome</td>
			<td style="width:159px;">1500</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Forceps</td>
			<td style="width:177px;">World Precision Instruments</td>
			<td style="width:159px;">555227F</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">100 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229693</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">35 mm petri dish</td>
			<td style="width:177px;">Cell treat</td>
			<td style="width:159px;">229638</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">50 ml falcon tube</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-432-22</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Woven nylon mesh 70 um</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">B0043D1SZG&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Micropipette</td>
			<td style="width:177px;">Gilson</td>
			<td style="width:159px;">F123602</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Glass pipette</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">NC-999363-9</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Aquarium sealant</td>
			<td style="width:177px;">Amazon.com, by MarineLand</td>
			<td style="width:159px;">Silicone Sealer 1 oz (Tube)&#160;</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Ring stand</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-675BO</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Microbore PTFE Tubing, 0.022&#34;ID</td>
			<td style="width:177px;">Cole-Parmer</td>
			<td style="width:159px;">WU-06417-21</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Modeling clay</td>
			<td style="width:177px;">Amazon.com</td>
			<td style="width:159px;">Sargent Art 22-4000</td>
			<td style="width:373px;">Any wax or oil based non-toxic modeling clay will suffice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Clamp</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">02-215-466</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">60ml syringe</td>
			<td style="width:177px;">Fisher</td>
			<td style="width:159px;">14-820-11</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;width:207px;">Embryo medium (E3)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">34.8 g NaCl 
			1.6 g KCl 
			5.8 g CaCl2&#183;2H2O 
			9.78 g MgCl2&#183;6H2O 
			 
			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.&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:207px;">Blocking Solution</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">50 ml TBS-NP-40 
			2.5 ml normal goat serum 
			1 g BSA 
			625 &#181;l Triton-X</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">TBS-NP-40 (pH 7.6)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">155 mM NaCl 
			10 mM Tris HCl 
			0.1% NP-40</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:207px;">2x MAB (pH6.4)</td>
			<td style="width:177px;"></td>
			<td style="width:159px;"></td>
			<td style="width:373px;">160 mM KPIPES 
			10 mM EGTA 
			2 mM MgCl2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:207px;">Commercial 3-D Image processing Software</td>
			<td style="width:177px;">PerkinElmer</td>
			<td style="width:159px;">Volocity (V 6.2)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dry block heater&#160;</td>
			<td style="width:177px;">VWR</td>
			<td style="width:159px;">12621-108</td>
			<td style="width:373px;">Used as a hot plate to melt agarose in Protocol 1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Dissecting Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">MZ12</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Confocal Microscope</td>
			<td style="width:177px;">Leica</td>
			<td style="width:159px;">SP5</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:207px;">Flat embedding mold</td>
			<td style="width:177px;">emsdiasum.com</td>
			<td style="width:159px;">BEEM 70904-01</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Public domain image processing software</td>
			<td style="width:177px;">NIH</td>
			<td style="width:159px;">ImageJ (V 1.5)</td>
			<td style="width:373px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:532px;">* 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 ...

Research

JoVE Journal

Developmental Biology

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

科研

发育生物学

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Microbead Implantation in the Zebrafish Embryo

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

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 ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

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 ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

两鲑鱼鱼、虹鳟 (虹鳟鱼虹鳟鱼) 和褐鳟 (鲑鲑形态石斑鱼) 的空气剥离效果

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...