Name: eye removal in living zebrafish larvae to examine innervation-dependent growth and development of the visual system
Uploaded: 2022-02-11T14:00:00
Description: Watch this Scientific Journal Video about Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System at JoVE.com

这项工作的资金主要由里德学院到KLC的启动资金，海伦斯塔福德研究奖学金基金到OLH，以及里德学院科学研究奖学金到YK。该项目始于史蒂夫威尔逊的实验室，与人力资源部门合作，人力资源部门得到了惠康信托学生奖学金（2009-2014）的支持。我们感谢Máté Varga，Steve Wilson和Wilson实验室的其他成员对这个项目的初步讨论，我们特别感谢Florencia Cavodeassi和Kate Edwards，他们是第一个教KLC如何在琼脂糖中安装胚胎并进行斑马鱼大脑解剖的人。我们还感谢Greta Glover和Jay Ewing帮助组装我们的钨针磨刀设备。

本文中的方法是根据里德学院和伦敦大学学院的机构动物护理和使用委员会的指南和批准的。有关本研究中使用的斑马鱼品系的详细信息，请参阅 材料表 。
1. 准备材料和工具
<ol><li>制定解决方案。<ol>
<li>通过将60x储备液（300mM NaCl，10.2mM KCl，20mM CaCl 2-二水合物和20mM MgCl2-六水合物）稀释在去离子水中，高压灭菌并在室温下储存来制?...

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本文中描述的技术说明了研究斑马鱼脊椎动物视觉系统发育的众多方法之一。其他研究人员已经发表了解剖胚胎视网膜并进行基因表达分析的方法19 或可视化视神经结构30中的神经元活动。本文提供了一种探索视网膜差异输入如何影响视神经组织中细胞行为的方法。
为了确保手术后成功消灭眼部和幼虫存活，重要的是幼虫健康，充分麻醉，...

该方法的目的是研究视网膜输入如何影响斑马鱼大脑视觉处理中心视神经技术的生长和发育。通过移除一只眼睛，然后比较视网膜的两侧，可以观察和归一化同一标本内的构造变化，从而能够比较多个标本。现代分子方法与该技术相结合，将深入了解视觉系统生长和发育以及轴突变性和再生的潜在机制。
感觉系统 - 视觉，听觉和躯体感觉 - 从外部器官收集信息并将该信息传递给中枢神经系统，生成中脑外部世界的“地图”1，2。视觉是几乎所有脊椎动物（包括许多鱼类）的主要感觉方式。视网膜是眼睛中的神经组织，它通过主要由光感受器，双极细胞和视网膜神经节细胞（RGC）组成的神经元回路收集信息，RGC是视网膜的投射神经元。RGC具有长轴突，它们穿过视网膜的内表面到达视神经头，在那里它们束曲并一起穿过大脑，最终终止于背中脑的视觉处理中心。这种结构在鱼类和其他非哺乳动物脊椎动物中被称为视技术，并且与哺乳动物的上丘脑同源3。
视神经结构是背中脑中双侧对称的多层结构。在斑马鱼和大多数其他鱼类中，视神经的每个叶仅接收来自对侧眼的视觉输入，使得左视神经终止于右构造叶，右视神经终止于左构造叶4 （图1）。像哺乳动物的对应物，上丘脑一样，视神经构造将视觉信息与其他感官输入（包括试听和躯体感觉）集成在一起，控制视觉注意力和眼球运动（如扫视1，5，6）的变化。然而，与哺乳动物上丘脑不同，视神经膜不断从称为构造增殖区7的内侧和尾部边缘附近的特化干细胞位产生新的神经元和神经胶质细胞。在视神经结构和中枢神经系统其他区域维持增殖祖细胞在一定程度上有助于斑马鱼8中记录的显着再生能力。
先前对盲人或独眼鱼大脑的研究表明，视神经结构的大小与其接受的视网膜神经支配量成正比9，10，11。在成年洞穴鱼中，其眼睛在早期胚胎发生时退化，视神经构造明显小于密切相关的目击面鱼9。洞穴鱼眼变性可以通过在胚胎发生过程中用表层鱼的晶状体替换内源性晶状体来阻止。当这些独眼洞穴鱼被饲养到成年期时，神经支配的构造叶比未支配的构造叶9含有约10%的细胞。同样，在用化学处理孵育以在同一个体内产生不同大小的眼睛的幼虫基利鱼中，具有更多神经支配的构造体的一侧更大并且包含更多的神经元10。来自成年金鱼视神经压碎实验的证据表明，神经支配促进增殖，当神经支配被破坏时，构造细胞增殖减少11。
证实并扩展了这些经典研究，最近的几份报告提供了数据，表明响应神经支配的增殖至少部分受到BDNF-TrkB途径12，13的调节。关于视神经构造细胞生长和发育的许多悬而未决的问题仍然存在，包括发育中的感觉系统如何应对损伤和轴突变性，哪些细胞和分子信号使视网膜输入能够调节视神经构造的生长，当这些机制变得活跃时，以及神经支配相关的增殖和分化是否使视网膜及其靶组织能够协调生长速率并确保准确的视网膜学映射。此外，关于活动依赖性发育还有更大的问题，可以通过用外科手术方法询问斑马鱼视觉系统来解决，如下所述。
为了研究神经活动（特别是来自视觉输入）改变细胞存活和增殖的细胞和分子机制，所述方法直接比较单个斑马鱼幼虫内神经支配和去神经支配的构造叶（图1）。该方法允许记录视神经组织中RGC轴突变性，并确认有丝分裂细胞的数量与神经支配相关。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63509/63509fig01.jpg"> 图1：单侧取眼前后斑马鱼幼虫的草图。 （A）在解剖显微镜下观察的5 dpf幼虫的图纸。每个幼虫都嵌入低熔点琼脂糖中，并横向定向，然后使用带有锋利钩状尖端的钨针将眼睛朝上舀出（在本例中为左眼）。（B） A中描述的手术产生的9 dpf幼虫的背视图图。只有来自右眼的三个高度模式化的RGC轴突显示脱发并与左脑叶中的神经元连接。缩写：dpf = 受精后的天数;dps = 手术后天数;RGC = 视网膜神经节细胞。 <a href="https://www.jove.com/files/ftp_upload/63509/63509fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment and supplies:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breeding boxes</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning high vacuum grease</td>
			<td>Sigma or equivalent supplier</td>
			<td>Z273554</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks (125 mL)</td>
			<td></td>
			<td></td>
			<td>For making Marc&#39;s Modified Ringers (MMR) with antibiotics for post-surgery incubation</td>
		</tr>
		<tr>
			<td>Fine forceps - Dumont #5</td>
			<td>Fine Science Tools (FST)</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes</td>
			<td>DWK Lifescience</td>
			<td>63A53 &#38; 63A53WT</td>
			<td>For pipetting embryos and larvae</td>
		</tr>
		<tr>
			<td>Glass slides for microscopy</td>
			<td>VWR or equivalent supplier</td>
			<td>48311-703</td>
			<td>Standard glass microscope slides can be ordered from many different laboratory suppliers.</td>
		</tr>
		<tr>
			<td>Glassware including graduated bottles and graduated cylinders</td>
			<td></td>
			<td></td>
			<td>For making and storing solutions</td>
		</tr>
		<tr>
			<td>2-part epoxy resin</td>
			<td>ACE Hardware or other equivalent supplier of Gorilla Glue or equivalent</td>
			<td>0.85 oz syringe</td>
			<td>https://www.acehardware.com/departments/paint-and-supplies/tape-glues-and-adhesives/glues-and-epoxy/1590793</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube (1.7 mL)</td>
			<td>VWR or equivalent supplier</td>
			<td>22234-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel plated pin holder (17 cm length)</td>
			<td>Fine Science Tools (FST)</td>
			<td>26018-17</td>
			<td>To hold tungsten wire while sharpening and performing surgeries/dissections.</td>
		</tr>
		<tr>
			<td>Nylon mesh tea strainer or equivalent</td>
			<td>Ali Express or equivalent</td>
			<td></td>
			<td>For harvesting zebrafish eggs after spawning; https://www.aliexpress.com/item/1005002219569756.html</td>
		</tr>
		<tr>
			<td>Paper clip</td>
			<td></td>
			<td></td>
			<td>For Tungsten needle sharpening device.</td>
		</tr>
		<tr>
			<td>Petri dishes 100 mm</td>
			<td>Fischer Scientific or equivalent supplier</td>
			<td>50-190-0267</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mm</td>
			<td>Fischer Scientific or equivalent supplier</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette pump</td>
			<td>SP Bel-Art or equivalent</td>
			<td>F37898-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>Sigma</td>
			<td>909122</td>
			<td>For Tungsten needle sharpening device. Make a 10% w/v solution of KOH in the hood by adding pellets to deionized water.</td>
		</tr>
		<tr>
			<td>Power supply (variable voltage)</td>
			<td></td>
			<td></td>
			<td>For Tungsten needle sharpening device. Any power supply with variable voltage will work (even one used for gel electrophoresis).</td>
		</tr>
		<tr>
			<td>Sylgard 184 Elastomer kit</td>
			<td>Dow Corning</td>
			<td>3097358</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire (0.125 mm diameter)</td>
			<td>World Precision Instruments (WPI)</td>
			<td>TGW0515</td>
			<td>Sharpen to remove eye and dissect larvae.</td>
		</tr>
		<tr>
			<td>Variable temperature heat block</td>
			<td>The Lab Depot or equivalent supplier</td>
			<td>BSH1001 or BSH1002</td>
			<td>Set to 40-42 &#176;C ahead of experiments.</td>
		</tr>
		<tr>
			<td>Wide-mouth glass jar with lid (e.g., clean jam or salsa jar)</td>
			<td></td>
			<td></td>
			<td>For Tungsten needle sharpening device.</td>
		</tr>
		<tr>
			<td>Wires with alligator clip leads</td>
			<td></td>
			<td></td>
			<td>For Tungsten needle sharpening device.</td>
		</tr>
		<tr>
			<td>Microscopes:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Any type will work but having adjustable transmitted light on a mirrored base is preferred.</td>
		</tr>
		<tr>
			<td>Laser scanning confocal microscope</td>
			<td></td>
			<td></td>
			<td>High NA, 20-25x water dipping objective lens is recommended. 
			Microscope control and image capture software (Elements) is used here but any confocal microscope will work.</td>
		</tr>
		<tr>
			<td>Reagents for surgeries and dissections:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td>For Marc&#39;s Modified Ringers (MMR) and embryo medium (E3).</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H7006</td>
			<td>For Marc&#39;s Modified Ringers (MMR).</td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-050</td>
			<td>Make 1% in embryo medium (E3) or Marc&#39;s Modified Ringers (MMR).</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma</td>
			<td>1374248</td>
			<td>For embryo medium (E3).</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma</td>
			<td>M7506</td>
			<td>For Marc&#39;s Modified Ringers (MMR).</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td>Dilute 8% (w/v) stock with 2x concentrated PBS (diluted from 10x PBS stock).</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma</td>
			<td>P4333-20ML</td>
			<td>Dilute 1:100 in Marc&#39;s Modified Ringers.</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) tablets</td>
			<td>Diagnostic BioSystems</td>
			<td>DMR E404-01</td>
			<td>Make 10x stock in deionized water, autoclave and store at room temperature. Dilute to 1x working concentration.</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td>For Marc&#39;s Modified Ringers (MMR) and embryo medium (E3).</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td>For Marc&#39;s Modified Ringers (MMR) and embryo medium (E3).</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td>Make 10 M and use to adjust pH of MMR to 7.4.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine-S</td>
			<td>Pentair</td>
			<td>100G #TRS1</td>
			<td>Recipe: https://zfin.atlassian.net/wiki/spaces/prot/pages/362220023/TRICAINE</td>
		</tr>
		<tr>
			<td>Reagents for immunohistochemistry:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexafluor 568 tagged Secondary antibody to detect rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-11011</td>
			<td>Use at 1:500 dilution for wholemount immunohistochemistry.</td>
		</tr>
		<tr>
			<td>DAPI or ToPro3</td>
			<td>Invitrogen</td>
			<td>1306 or T3605</td>
			<td>Make up 1 mg/mL solutions in DMSO; 1:5,000 dilution for counterstaining.</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td>A component of immunoblock buffer.</td>
		</tr>
		<tr>
			<td>Methanol (MeOH)</td>
			<td>Sigma</td>
			<td>34860</td>
			<td>Mixing MeOH with aqueous solutions like PBST is exothermic. Make the MeOH/PBST solutions at least several hours ahead of time or cool them on ice before using.</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>ThermoFisher Scientific</td>
			<td>50-062Z</td>
			<td>A component of immunoblock buffer. Can be aliquoted in 1-10 mL volumes and stored at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Primary antibody to detect phosphohistone H3</td>
			<td>Millipore</td>
			<td>06-570</td>
			<td>Use at 1:300 dilution for wholemount immunohistochemistry.</td>
		</tr>
		<tr>
			<td>Primary antibody to detect Red Fluorescent Protein (RFP; detects dsRed derivatives)</td>
			<td>MBL International</td>
			<td>PM005</td>
			<td>Use at 1:500 dilution for wholemount immunohistochemistry.</td>
		</tr>
		<tr>
			<td>Proteinase K (PK)</td>
			<td>Sigma</td>
			<td>P2308-10MG</td>
			<td>Make up 10 mg/mL stock solutions in PBS and use at 10 &#181;g/mL.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Useful to make a 20% (v/v) stock solution in PBS.</td>
		</tr>
		<tr>
			<td>Software for data analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ (Fiji)</td>
			<td></td>
			<td></td>
			<td>freeware for image analysis; https://imagej.net/software/fiji/</td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td></td>
			<td></td>
			<td>freeware for statistical analysis and data visualization; https://www.rstudio.com/products/rstudio/download/</td>
		</tr>
		<tr>
			<td>Adobe Photoshop or GIMP</td>
			<td></td>
			<td></td>
			<td>Proprietary image processing software (Adobe Photoshop and Illustrator) are often used to compose figures). A freeware alternative is Gnu Image Manipulation Program (GIMP; https://www.gimp.org/)</td>
		</tr>
		<tr>
			<td>Zebrafish strains</td>
			<td></td>
			<td></td>
			<td>available from the&#160; Zebrafish International Resource Centers in the US (https://zebrafish.org/home/guide.php) or in Europe (https://www.ezrc.kit.edu/). Specialized transgenic strains that have not yet been deposited in either resource center can be requested from individual labs after publication.</td>
		</tr>
	</tbody>
</table>

eye removal in living zebrafish larvae to examine innervation-dependent growth and development of the visual system

斑马鱼表现出非凡的终身生长和再生能力。例如，在胚胎发生期间建立的专用干细胞生态位支持整个视觉系统在眼睛和大脑中的连续生长。视网膜和视神经结构之间的协调生长确保了在眼睛和大脑中添加新神经元时准确的视网膜图谱图。为了解决视网膜轴突是否为调节颅骨干和祖细胞行为（如存活、增殖和/或分化）提供关键信息，有必要能够比较同一动物内和动物之间的神经支配和去神经支配的构造叶。 
手术从活的幼虫斑马鱼中切除一只眼睛，然后观察视网膜，实现了这一目标。随附的视频演示了如何麻醉幼虫，电解磨钨针，并用它们去除一只眼睛。接下来，它展示了如何从固定的斑马鱼幼虫中解剖大脑。最后，该视频概述了免疫组织化学方案，并演示了如何在低熔点琼脂糖中安装染色胚胎以进行显微镜检查。

为了确认眼睛切除是否完整并评估视神经结构如何变化，在 Tg[atoh7：RFP] 菌株中进行了手术，该菌株用膜靶向RFP标记所有RGC，从而标记从视网膜投射并形成视神经的所有轴突24。虽然使用这种菌株不是绝对必要的，但它可以直接观察和可视化视神经桥神经瘘中的视神经末端。标记视神经的其他方法，例如用亲脂性DiI或DiO染料25，26...

Watch this Scientific Journal Video about Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System at JoVE.com

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

本文解释了如何手术从活斑马鱼幼虫身上摘除眼睛，作为研究视网膜输入如何影响视神经结构生长和发育的第一步。此外，本文还提供了有关幼虫麻醉、固定和脑解剖的信息，然后是免疫组化和共聚焦成像。

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

所以我的实验室有兴趣了解眼睛和大脑是如何一起生长的。这项技术使我们能够研究视网膜输入如何影响大脑的生长和发育。手术从活的幼虫斑马鱼身上切除一只眼睛，然后观察视神经结构，使我们能够比较同一动物内和动物之间的神经支配和去神经支配的构造叶。将这种技术与现代分子方法相结合，可以为神经发育，再生和退化背后的机制提供新的见解。首先，将阴极和阳极线连接到电源。将阴极线末端的鳄鱼夹连接到部分拉直的回形针上，然后将回形针插入氢氧化钾溶液中，将其连接到罐子的侧面。将阳极线的鳄鱼夹连接到针座的颈部。将功率调至约20伏，并将钨丝浸入氢氧化钾溶液中，以一定角度将导线从溶液中拉出，以电解方式将导线磨成细尖。在解剖显微镜下检查针尖，以确保其足够锋利。当尖锐的针头通过轻轻触摸培养皿来弯曲时，会产生弯曲针尖。在手术当天，使用宽口径玻璃牧场移液器将10至15只幼虫转移到装满新鲜E3的35毫米培养皿中。向幼虫中加入三到五滴麻醉剂。为了确定幼虫是否得到充分的麻醉，请寻找缺乏触摸反应。如果幼虫在三分钟后对触摸仍然有反应，再添加两到三滴麻醉剂，然后重新评估。为了固定幼虫进行手术，将一只幼虫带到只有少量E3的窄口径玻璃牧场移液器中。接下来，将约200微升熔化的1%低熔点或LMP琼脂糖放入含有幼虫的移液器中并混合，以确保幼虫悬浮在LMP琼脂糖中。将35毫米培养皿的盖子面朝上放在解剖显微镜下，然后将幼虫和琼脂糖喷射到倒置的培养皿盖上。传播琼脂糖，使幼虫位于液滴的中心。使用钝钨针，快速但轻轻地操纵幼虫，使其横向，一只眼睛朝上。等待几分钟，让琼脂糖凝固。沿着眼眶的边缘，使用精细电解磨的钨针的尖端刺穿眼睛周围的皮肤。然后从眼睛的颞腹侧将针的边缘滑入眼睛下方。使用受控压力将眼睛从眼窝中释放出来。继续用针的一侧在背侧和前部按压眼睛，最终穿过视神经并释放眼睛。或者，您可以使用非常精细的手术镊子通过捏住视神经并将眼睛从内侧推到外侧来移除眼睛。成功去除眼睛后，用MMR溶液覆盖琼脂糖，通过将钨针轻轻刷在头部周围然后在其身体周围，同时用镊子稳定培养皿盖，将每个幼虫从琼脂糖中释放出来。手术后，将幼虫置于补充有抗生素的MMR溶液中，直到第二天，然后将幼虫返回到E3并将它们饲养到实验的终点。幼虫绝症麻醉后，用4%多聚甲醛固定。将固定的幼虫悬浮在解剖显微镜下的密封板上的PBS液滴中。通过将两个钨针穿过套管来横向固定它们，其中一个引脚位于覆盖AGM区域的着色区域后面，另一个引脚与蛋黄延伸的末端对齐。用锋利的钨针和细镊子取下眼睛。有时可以将针头穿过下颌戳到另一侧，然后取出另一只眼睛。使用钨针从耳朵的颞侧到腹侧划伤。在运动中的相同动作中，将针头后部带到下颌，轻轻地向前拉动，直到耳朵和下颌被移除。通过将针头戳入下颌来移除另一只眼睛和耳朵。使用镊子拉出腹侧器官和剩余的蛋黄。最后，在后脑和脊髓交界处附近的背颅皮肤上做一个浅切口。用镊子抬起皮肤，将其拉至端脑前部和周围。用镊子去除任何剩余的组织。解开幼虫并转移到PBS中放入1.5毫升微量离心管中。用真空润滑脂将腔室载玻片固定在100毫米培养皿的盖子中。将免疫染色和加工的幼虫转移到孔板或凹陷载玻片上，用解剖显微镜观察它们。将幼虫安装在1%LMP琼脂糖柱中的腔室载玻片上。使用玻璃经过移液器，将一个幼虫放在腔室载玻片上，尽可能少地沉积PBS。使用相同的移液管用融化和温热的1%LMP琼脂糖覆盖幼虫。将LMP琼脂糖移液到柱中，然后尽可能对称地定位幼虫，背表面可见。切除眼睛后，在视神经鞘中观察到视网膜轴突的进行性变性。手术后两天，RFP标记的轴突表现出快速瓦勒变性的标志，例如炙炙和碎片化。如箭头所示，在构造器右侧的神经疙内外均注意到RFP阳性点状物。到手术后四天，右颅叶的碎片轴突和RFP标记的轴突碎片显着减少，表明死亡和退化轴突的清除相对较快。通过解剖皮肤和结缔组织的眼睛，下巴，耳朵和颅骨来暴露大脑。理想情况下，大脑在这个过程中保持完整。然而，当皮肤或下颌的颅盖被移除时，前脑的某些部分，特别是嗅球，可能会受损或完全切除。此外，有时在刺穿或捏住皮肤以将其从大脑中拉出时，构造的侧壁被切开。没有两个样本是完全相同的。因此，您需要能够根据需要通过切除眼睛和大脑解剖来做出微妙的调整。患者和锋利的工具是成功的关键。该技术可以通过细胞和分子方法进行跟进，例如体内活细胞成像或RNA测序，以获得对视神经结构生长和发育的分子和细胞机制的新见解。

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

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 ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

多光子时间推移成像实时可视化发展： 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

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

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Whole Mount Immunohistochemistry in 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. ...