作者要感谢大卫 Langenau 慷慨地提供水族馆空间;埃里克石头, 约翰 c. 摩尔和秦唐帮助与斑马鱼的维护和试剂, 和安妮罗伯逊和艾略特 Hagedorn 从伦纳德区的实验室购买的斑马鱼菌株在这里使用。他们还要感谢奥克塔维奥 Hurtado 关于光刻技术的建议。FE 的经费来自实力的儿童医院和美国澳大利亚协会的研究金。这项工作由 NIH 资助 GM92804。

根据《2011N000127 议定书》, 马萨诸塞州总医院研究动物保育小组委员会批准了微注射幼虫。
1. 设备制造
注: 此处描述的用于设计光刻掩模的所有计算机辅助绘图 (CAD) 文件 (图 1) 均可供下载。查看链接的材料表。
<ol><li>使用标准光刻技术7, 在1000级洁净室中制造主模具晶片。对于微结构阵列和通道, 按照制造商的说明, 在3层中依次使用3个单独的掩码对环氧基负光刻胶进行模式设计: 100 µm 为浅特征, 200 µm 为中等特征, 400 µm的深层特征。对于成像设备, 使用400µm 为浅层特征和600µm 的深部特征模式两个图层。</li>
	<li>将完成的晶片带到 15 cm 培养皿的底座上 (图 2)。</li>
</ol>2. 微表面阵列的制备
<ol><li>制作可用的设备, 铸造烷 (矽), 使用主晶片作为模具。将50克的硅烷单体与5克的引发剂结合在一起, 在塑料称重皿中彻底混合。</li>
	<li>把混合物仔细地浇在模子上。</li>
	<li>德加在真空干燥在 16-25 inHg (54-85 帕斯卡) 为 1 h。</li>
	<li>为了治疗, 在65° c 烘烤至少3小时。该公司应治疗的坚定, 但灵活的固体。</li>
	<li>小心地用手术刀切割主晶片上的装置, 确保手术刀尖端与晶片表面保持接触。使用镊子, 剥离的硅烷副本远离硅片和转移到一个干净的 10 cm 培养皿, 功能侧向上 (图 3)。</li>
	<li>三十五年代用等离子炉对该装置进行氧等离子体处理, 以减少憎水性。</li>
	<li>盖有斑马鱼胚胎培养基 (E3), 含168毫克/升的乙基 3-aminobenoate 甲磺酸 (MS-222, pH 7.5)。使用转移吸管从设备表面流出的水, 从更深的特征中除去任何气泡。</li>
</ol>3. 微表面阵列的制备-琼脂糖
<ol><li>要制作一个负的硅橡胶模具, 首先要处理的是一个具有氧等离子体的硅橡胶装置。</li>
	<li>
使用 1h、1h、2h、2hPerfluorooctyltriethoxysilane (PFDTS) 硅烷蒸气来创建一个惰性表面8。简要:
	<ol>
<li>在通风柜内的真空室中, 将待处理的硅橡胶置于特征侧。</li>
		<li>在µL 旁边, 放置一个小的开放的玻璃或铝盘, 小心翼翼地将200的 PFDTS (98% 硅烷) 移入盘中。 注意: PFDTS 硅烷是高度挥发性的, 与水剧烈反应, 是易燃的, 在接触时会引起严重的皮肤和眼部损伤。使用前请仔细阅读 MSDS。</li>
		<li>关闭真空室, 并应用真空15-30 分钟, 或直到 PFDTS 硅烷完全蒸发。</li>
	</ol>
</li>
	<li>将该设备放置在培养皿中, 并将其用作新的硅橡胶的模具, 如步骤 2.1-2.3 所述。</li>
	<li>在65° c 烘烤至少3小时, 然后从处理过的装置和新鲜的培养皿中剥离整个新鲜的硅氧烷层。这可以用来作为一个负模铸造琼脂糖。</li>
	<li>准备琼脂糖, 煮沸2% 溶液的低凝胶温度琼脂糖在 E3 在微波炉, 直到琼脂糖完全溶解。</li>
	<li>倒入热的琼脂糖的消极模具和删除任何气泡的琼脂糖覆盖设备的流动热琼脂的功能与转移吸管。</li>
	<li>一旦气泡被去除, 冷藏在4° c 设置琼脂糖。</li>
	<li>通过手工将 E3 从下方变形, 将琼脂糖块转移到新的培养皿中, 然后用含有 MS-222 的含水的湿剂把琼脂糖块从底片上取下。</li>
</ol>4. 制备硅橡胶成像器件
<ol><li>制作可使用的设备, 铸造硅晶片, 用主晶圆作为模具。将50克的硅烷单体与5克的引发剂 (与步骤2.1 相同) 结合在一起, 用塑料叉将塑料称量盘彻底混合。</li>
	<li>把混合物仔细地浇在模子上。</li>
	<li>德加在真空干燥在 16-25 inHg (54-85 帕斯卡) 为 1 h。</li>
	<li>为了治疗, 在65° c 烘烤至少3小时。该公司应治疗的坚定, 但灵活的固体。</li>
	<li>从主晶片切割设备, 用 1.5 mm 冲床冲出端口。</li>
	<li>如步骤2.6 所述, 三十五年代用氧等离子体处理设备和6井玻璃底井板。</li>
	<li>债券一设备的特点-侧向下到每个玻璃片的 6-井板通过放置在一个85° c 板10分钟。 注: 为了确认粘合成功, 使用镊子将牢固的压力施加到粘合装置的一侧。设备应保持在玻璃片。</li>
</ol>5. 斑马鱼文化
<ol><li>养殖斑马鱼胚胎到 2 dpf 使用标准技术3。</li>
	<li>Dechorionate 胚胎使用钳或1毫克/毫升蛋白酶混合 (见材料表)3至少30-60 分钟之前加载到设备, 让他们伸直。</li>
	<li>麻醉胚胎使用 (168 毫克/升) MS-222 (pH 7.5), 加入1毫升的25X 库存解决方案的 E3 在其培养皿。</li>
</ol>6. 斑马鱼对微表面-草皮阵列的定位
<ol><li>在其培养皿中准备2.7 步 (图 3A) 中的硅橡胶块, 使其覆盖一层薄薄的 (1-2 毫米) 的 E3, 从而更容易操作胚胎。</li>
	<li>使用转移吸管将所需的胚胎数量 (一般为10-20 每个条件) 转移到硅橡胶块的表面。</li>
	<li>使用微工具 (发圈或类似的), 将胚胎推入微草皮 (图 3A)。草皮的使用特别适合背部和腹侧方向。 注: 对于草皮与更紧的适合 (背部和腹), 尝试定位的胚胎在块的表面平行的草皮, 然后滚动到位置使用头发循环。把他们推向更深的草皮将有助于保持他们的稳定。</li>
</ol>7. 斑马鱼在微表面-微通道上的定位
<ol><li>使用转移吸管绘制 E3 的微通道 (图 3B), 直到 E3 的水平低于该块的边缘, 但仍存在于储层和通道中, 并位于该层的一个薄薄的图层上。</li>
	<li>使用转移吸管将10胚胎从步骤5.3 转移到水库, 注意不要溢出边缘。</li>
	<li>使用头发循环, 操作每个胚胎进入漏斗在适当的渠道入口和东方, 以便胚胎的头朝向渠道, 并且胚胎有适当的取向, 当它进入渠道。</li>
	<li>使用毛发循环将每个胚胎向下滑动, 直到它到达通道壁上的定向 microfeatures, 帮助保持它的位置。使用微通道是特别推荐的横向方向。 注: 为了减少微注射过程中的胚胎滑动, 请通过从储层中提取 E3 来调整表面张力的大小。</li>
</ol>8. 斑马鱼的显微注射
<ol><li>
准备注射针头。
	<ol>
<li>使用微拉拔器制作硼硅酸盐玻璃 microcapillary 针, 如前所述4。所产生的针应该是锥形到一个点在一端, 与锥形长度约10毫米。</li>
		<li>准备注射的溶液, 在这种情况下, 100 nm 趋化剂 (n-Formylmethionine-亮-苯丙氨酸 (fMLP) 或烯 B4 (LTB4)) 和 100 nm 70 kDa 罗丹明葡聚糖在 PBS 中的示踪物。</li>
		<li>使用一个细锥形吸管尖端 (见材料表) 将5µL 的溶液装入针中。</li>
		<li>将针安装在磁性支架上的微上, 并连接到 picopump 微量。</li>
	</ol>
</li>
	<li>用镊子捏锥形尖端, 在45°角处折断针尖。</li>
	<li>通过调整 picopump 控制器上的注入时间, 调整注塑丸大小为 1 nL。</li>
	<li>调整注射针头的角度。在微控制器上, 45 °的针角通常是一个好的起点, 针与胚胎的长度平行。一个较陡的角度可以用来最小化胚胎在显微注射过程中的横向运动。</li>
	<li>用左手和针微控制培养皿, 使针尽可能靠近注射的目的部位, 在这种情况下, 耳囊泡。 注: 耳囊可被确定为眼睛后部的长方形器官, 有两个较小的不同的暗长方形结构 (耳)。</li>
	<li>使用微控制器, 穿透靶组织 (在这种情况下, 耳泡), 使针尖的针头是在囊泡, 并注入预设的音量使用 picopump 脚开关。 注意: 如果组织抵抗渗透, 试着轻轻地敲击微控制器旋钮。</li>
	<li>为了释放胚胎, 通过转移吸管从上到下的表面流动 E3, 或者旋转盘状, 使胚胎被释放到周围的 E3。</li>
	<li>使用转移吸管将胚胎移植到 E3 无 MS-222 的恢复盘中。</li>
</ol>9. 使用硅橡胶成像装置对胚胎进行筛选
<ol><li>从步骤4.7 中的 E3 (+ MS-222) 通过端口来进行设备的质数。这可以用1000µL 吸管或窄尖端转移吸管来完成。</li>
	<li>用 E3 + MS-222 填充, 直到设备盖好。</li>
	<li>使用转移吸管从步骤8.7 向每个井输送4注入的胚胎。胚胎可以预先麻醉, 如果需要孵化在 E3 + MS-222 在1-2 分钟之前加载 (步骤 5.3)。</li>
	<li>使用微工具 (发圈或类似的), 将胚胎置于成像通道入口的方向 (根据所使用的设备), 将其定位到所需的位置 (尾部首先或头部), 并将它们部分推入设备 (图 4A)。这将有助于他们均匀地进入设备。</li>
	<li>使用1000µL 吸管或转移吸管从端口绘制 E3, 直到将胚胎以正确的成像方向绘制到通道中 (图 4B)。 注意: 小心不要把胚胎从诱捕点吸出来, 这样会破坏胚胎并改变它们的方向。</li>
	<li>将井板转移到显微镜进行成像 (图 4C)。</li>
	<li>
图像使用倒置荧光显微镜。
	<ol>
<li>通过选择适当的镜头来调整放大倍数。10X 通常是一个有用的放大率成像斑马鱼中性粒细胞迁移。</li>
		<li>调整每个通道的光源强度和曝光时间, 使每个信号清晰, 但不饱和。</li>
		<li>捕获每个通道的图像。在这里, 我们使用绿色荧光蛋白 (GFP, 前: 470/22, Em: 525/50), 得克萨斯州红 (TxRed, 前: 585/29, Em: 628/32) 和传输通道。在后处理过程中可以组合多通道图像 (图 4B-I)。</li>
	</ol>
</li>
</ol>

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R., Wilson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+in+a+microfluidic+format+using+flexible+and+compliant+channels+and+electroosmotic+dosage+control.">Microinjection in a microfluidic format using flexible and compliant channels and electroosmotic dosage control.</a> Lab Chip. 9 (22), 3202-3211 (2009).</li></ol>

作者声明没有利益冲突。

在这里, 我们描述了使用的设备, 我们最近开发, 以方便 2 dpf 斑马鱼微注射5, 并介绍了一个简单的琼脂糖免费安装装置, 方便成像的胚胎。这些工具突出了光刻技术的效用, 用于制作适用于斑马鱼技术的设备。
我们发现 MSA 装置特别适用于注射针内的细胞或微粒, 如真菌孢子或人类癌细胞, 这需要使用更大的钻孔针来运送。注射到耳泡 (图 4</strong...

斑马鱼已经成为一个强大的模型, 许多领域, 从基础发展生物学研究到大规模的遗传和化学屏幕1,2。常规的基因操作, 如基因过度表达、击倒、CRISPR/Cas9 突变和转基因依赖于单细胞受精卵的微注射, 这导致了简单易用的商业用于定向和稳定注射的卵子的可用工具3。其他方法, 如移植和感染, 往往需要注射到后期胚胎和幼虫使用较大的规范毛细管针头4。然而, 使用更大的规格针提出了重大的技术挑战, 因为它是更难穿透目标组织不推或滚动的胚胎。在这些条件下, 获得稳定胚胎所需的适当的水张力, 同时避免在手术过程中干燥是困难的, 而且胚胎可能不是理想的定向注射到靶组织。
在注射后, 筛出的胚胎选择那些已成功注射的胚, 并捕捉初始时间点的图像, 通常是有用的。为了应对这些挑战, 我们开发了一系列的微设备, 帮助稳定 2 dpf 胚胎的各种方向, 既用于微注射5, 也用于快速基于图像的扫描后注射。
为了在这些装置中获得足够的结构分辨率, 我们利用了光刻技术。这些方法通常用于微电子工业, 并且最近被外推用于微流控芯片的制造, 因此可以实现垂直结构, 范围从1-1、000µm, 这是一种适合于斑马鱼胚胎和幼虫操作的鳞片。所有的设备都是用烷 (硅氧烷) 制造的, 它价格低廉, 身体健壮, 生物惰性, 而且透明。
微表面阵列 (协议) 被格式化成块的, 具有图案的顶面, 类似于简单的渠道琼脂糖块通常用于卵注射。对于后注射筛选, 6 成像设备可以排列在一个标准的玻璃底 6-井板。这些装置是为易于装载的胚胎而设计的, 而卸载程序方便地允许对特定的胚胎进行抢救, 比以前由毕比实验室6。

<table>
	<tbody>
		<tr>
			<td>Dow Corning Sylgard 184&#160; Polydimethylsiloxane (PDMS)&#160;</td>
			<td>Ellsworth Adhsives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>For casting the devices. Kit includes PDMS monomer and Initiator</td>
		</tr>
		<tr>
			<td>Low gelling temperature agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9414-10G</td>
			<td>For casting agarose devices</td>
		</tr>
		<tr>
			<td>PFDTS silane</td>
			<td>Sigma Aldrich</td>
			<td>448931-10G</td>
			<td>For casting of negative PDMS molds</td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma Aldrich</td>
			<td>E10521-10G</td>
			<td>To anesthetize&#160; zebrafish&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine Dextran 70,000 Da</td>
			<td>ThermoFisher</td>
			<td>D1818</td>
			<td>To trace microinjections</td>
		</tr>
		<tr>
			<td>Leukotriene B4 (LTB4)</td>
			<td>Cayman Chemicals</td>
			<td>20110</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>N-Formylmethionine-leucyl-phenylalanine (fMLP)</td>
			<td>Sigma Aldrich</td>
			<td>F3506-50MG</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>15 cm Petri dish</td>
			<td>Fisher scientific</td>
			<td>08-757-148</td>
			<td>For Casting from the master wafer</td>
		</tr>
		<tr>
			<td>Glass-bottom 6-well plates</td>
			<td>MatTek</td>
			<td>P06G-0-20-F</td>
			<td>For imaging devices</td>
		</tr>
		<tr>
			<td>Borosilicate glass microcapillaries</td>
			<td>World Scientific Instruments</td>
			<td>TW-100-4</td>
			<td>For microinjection needles</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sigma Aldrich</td>
			<td>Z350796</td>
			<td>For transferring zebrafish embryos</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Fisher scientific</td>
			<td>E5242956003</td>
			<td>For loading the microinjection needles</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 1.5 mm punch</td>
			<td>Ted Pella Inc.</td>
			<td>15111-15</td>
			<td>To punch ports in PDMS imaging devices</td>
		</tr>
		<tr>
			<td>No.&#160;11 Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For cutting PDMS&#160;</td>
		</tr>
		<tr>
			<td>Dumont No. 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-10</td>
			<td>For dechorionating embryos and breaking microinjection needle tips</td>
		</tr>
		<tr>
			<td>Marzhauser Micromanipulator</td>
			<td>ASI&#160;</td>
			<td>MM33-R</td>
			<td>For manipulating microinjection needle</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>MSC</td>
			<td>SPI - 87242624</td>
			<td>For mounting micromanipulator</td>
		</tr>
		<tr>
			<td>MPPI-3 Picopump controller</td>
			<td>ASI</td>
			<td>MPPI-3</td>
			<td>To control microinjection volume and timing</td>
		</tr>
		<tr>
			<td>EVOS inverted fluorescent microscope</td>
			<td>ThermoFisher</td>
			<td>EVOS FL</td>
			<td>To image injected embryos</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>For visualizing microinjecion</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk</td>
			<td></td>
			<td>Download AutoCAD files from: https://dx.doi.org/10.6084/m9.figshare.4282853 and on the ZFIN community protocols wiki page: https://wiki.zfin.org/display/prot/ZFIN+ Protocol+Wiki &#160;</td>
		</tr>
	</tbody>
</table>

microstructured devices for optimized microinjection and imaging of zebrafish larvae

斑马鱼已经成为各种人类疾病的强有力的模型, 并成为越来越多的实验研究的有用工具, 通过大规模的遗传和化学屏障跨越了基础发育生物学。然而, 许多实验, 特别是那些与感染和异种模型有关的试验, 依赖于胚胎和幼虫的显微注射和成像, 这些都是需要技能和专长的费力技术。为了提高目前显微注射技术的精确度和吞吐量, 我们开发了一系列微装置, 在2天后, 在腹侧、背部或侧面定向后, 对斑马鱼胚胎进行定位和稳定。程序.为了帮助对胚胎进行成像, 我们还设计了一个简单的装置, 使4只斑马鱼在平行的方向上与一个玻璃覆盖层滑动。在这里, 我们展示了光刻方法的有效性, 为斑马鱼技术的优化创造了有用的设备。

此处介绍的方法演示了设计 (图 1) 和用于 2 dpf 斑马鱼的设备的制造, 使用光刻 (图 2) 和软光刻 (图 3) 技术。此方法允许对许多设计迭代和修改进行快速测试, 并且在其他开发阶段与斑马鱼一起使用的微观结构尺寸的改变和优化可能会延长它们的应用范围。
我们...

Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

斑马鱼胚胎和幼虫的显微注射是许多斑马鱼模型中的关键但具有挑战性的技术。在这里, 我们提出了一系列的微型工具, 以帮助稳定和定位斑马鱼的显微注射和成像。

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

microstructured-devices-for-optimized-microinjection-imaging

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.5K 次观看.  Massachusetts General Hospital–Harvard Medical School–Shriners Burns Hospital. 斑马鱼胚胎和幼虫的显微注射是许多斑马鱼模型中的关键但具有挑战性的技术。在这里, 我们提出了一系列的微型工具, 以帮助稳定和定位斑马鱼的显微注射和成像。

视频: Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

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

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 ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

斑马鱼发展长期成像的多功能安装方法

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

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. ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

原位 斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the specific effects and mechanisms in zebrafish remain unclear. Alizarin red has a high affinity for calcium ions and can help visualize the bone and illustrate skeletal mineral mass. In this study, we aimed to detect lead acetate (PbAc)-induced bone loss in zebrafish larvae by using alizarin red staining. Zebrafish embryos were treated with a series of PbAc concentrations (0, 5, 10, 20 mg/L) between 2 and 120 h post fertilization. Whole-mount skeletal staining was conducted on larvae at 9 days post fertilization, and the total stained area was quantified using ImageJ software. The results indicated that the mineralized tissues were stained in red, and the stained area decreased significantly in the PbAc-exposure group, with a dose-dependent change in bone mineralization. This paper presents a staining protocol for investigating skeletal changes in PbAc-induced bone defects. The method can also be used in zebrafish larvae for the detection of bone loss induced by other chemicals.

Here, we have used alizarin red staining to show that lead acetate exposure causes a bone mass change in zebrafish larvae. This staining method can be adapted to the investigation of bone loss in zebrafish larvae loss induced by other hazardous toxicants.

使用茜素红染色检测斑马鱼幼虫中化学诱导的骨质流失

Chemically induced bone loss due to lead (Pb) exposure could trigger an array of adverse impacts on both human and animal skeletal systems. However, the ...

灵长类动物大脑类器官的基因改造

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

作者聚焦:灵长类动物脑类器官的靶向显微注射和电穿孔以进行基因改造  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

灵长类动物脑类器官的靶向显微注射和电穿孔用于基因修饰

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

斑马鱼幼虫显微注射和成像的微装置

摘要

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此视频中的章节

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

在活斑马鱼胚胎成像的亚细胞结构

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

显微注射转基因的基因组，并在编辑Threespine刺鱼

斑马鱼发展长期成像的多功能安装方法

斑马鱼胚胎和幼虫的整个山免疫性化学

原位斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交

使用茜素红染色检测斑马鱼幼虫中化学诱导的骨质流失

灵长类动物脑类器官的靶向显微注射和电穿孔用于基因修饰

斑马鱼幼虫显微注射和成像的微装置

摘要

探索更多视频

此视频中的章节

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

在活斑马鱼胚胎成像的亚细胞结构

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

显微注射转基因的基因组，并在编辑Threespine刺鱼

斑马鱼发展长期成像的多功能安装方法

斑马鱼胚胎和幼虫的整个山免疫性化学

原位 斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交

使用茜素红染色检测斑马鱼幼虫中化学诱导的骨质流失

灵长类动物脑类器官的靶向显微注射和电穿孔用于基因修饰

原位斑马鱼幼体和幼鱼在雌雄同体发育过程中的杂交