We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

该协议遵循了德国和柏林国家法律的动物护理准则;斑马鱼的处理是由动物保护（LaGeSo，柏林 - 勃兰登堡）地方当局监控。 1.获取斑马鱼胚胎的心脏提取<ol><li>横心脏记者斑马鱼，例如作为Tg（MYL7:EGFP）转基因twu34线9，以便获得胚胎心脏特异性GFP表达。 </li><li>保持水蛋胚胎在28.5°C，直到所需的萌芽阶段10,11。确保所收集的胚胎人口是同质既遗传和发育阶段以限制的变化的基因表达。 </li><li>手动Dechorionate胚胎。 </li></ol> 2.解剖斑马鱼胚胎的心脏注意:保留所有的解决方案上的冰。如果可能的话，做团队:一个人从教统局解剖的心ryos而另一个人在排序的荧光体视显微镜的心。这允许几百胚胎在几个小时的处理。 <ol><li>转约100胚胎成1.5ml的离心管中。麻醉用三卡因的胚胎（0.16毫克/毫升在E3的培养基）。一旦进入胚胎显然麻醉（他们不游泳和沉积物的试管底部，但他们的心还在跳动）。 </li><li>除去三卡因/ E3溶液和1毫升的L-15/10％FBS培养基中，一旦洗涤胚胎并在冰上保持。 注:L-15/10％FBS培养基中是很重要的，整个解剖过程中，以保持器官和细胞存活，直到心已被转移到RNAlater中或Trizol试剂。 </li><li>加入1毫升的L-15/10％FBS培养基中的胚胎和移液器上下用圆形凝胶加载提示5-8次，直到蛋黄被完全破坏。吸取胚胎逐滴到溶液的表面上，从而使压降将会破裂和将胚轻轻破碎。 注意:如何大力和频率胚胎必须吸取上下取决于胚胎的发育阶段， 即多移液需要在后期（ 例如在56 HPF）。 </li><li>对于第一批解剖胚胎，评估胚的完整性并解剖心在立体显微镜下的比例，因为一些心中可保持附着在胚胎如果过轻轻吸入。调整移液的方式为下轮清扫相应。 注:使用低滞留枪头和离心管，以减少心中坚持的塑料。 </li><li>样品应用到100微米的过滤器放置在50毫升离心管中;冲洗1.5 ml管1毫升的L-15/10％FBS培养基中，然后将此到滤波器，以便最小化样品的损失（一些心可能被粘在管的壁）。 1毫升L洗两次过滤器-15 / 10％FBS中。在此步骤中心通过过滤器，并收集在50ml管中。 </li><li>应用流通到30微米的过滤器放置在一个15毫升的离心管中，用1ml的L-15/10％FBS培养基冲洗过滤器一次。在这第二个过滤步骤中，心被保留在过滤器和较小的碎片被洗掉。 </li><li>转动过滤器倒置并通过施加连续洗涤三次用1ml的L-15/10％FBS冲洗心中从过滤器的成在1％琼脂糖包被的培养皿。 </li><li>手动分离GFP阳性的心从非荧光胚胎碎片（ 如眼镜头）与一对钳下，荧光体视显微镜，集中他们的菜心。根据步骤3收集它们。 注:如果胚胎是年龄超过24 HPF，心中应跳动，表明组织仍然生理正常。 </li></ol> 3.从Dissec分离mRNA特德心<ol><li>收集在尽可能小的体积的心（ 例如 10微升），并吸移管到1.5ml试管含有0.75毫升RNAlater中（在冰上）。确认没有心留在枪头通过查看它在荧光体视显微镜。 </li><li>或者，如果一种化学罩是可以在荧光显微镜的附近，在化学罩所收集的心转移到的Trizol（警告）。这就避免心贴在了枪头，也是心灵的离心过程中可能的损失（步骤3.4，3.5和3.7）。汇集来自几轮孤立的心在同一个管的Trizol，并直接转到步骤3.8;最终的体积应不超过原始的Trizol体积的10％。 注意:请阅读的Trizol材料安全数据表（MSDS）后再使用。处理Trizol试剂罩下，并建议穿戴个人防护装备。避免与皮肤，眼睛和clothi接触纳克。去除所有火源。 </li><li>普尔从几轮隔离心中到相同的管中的RNAlater（在冰上），与组织的收集量的RNA分离的效率提高。如果总样品体积超过原始RNAlater中体积的10％，可以使用一个额外的管0.75毫升RNAlater中存储（在冰上）。 </li><li>离心样品在15700 XG为20分钟，在4°C至沉淀的心。 </li><li>下一个荧光立体显微镜，收集仔细尽可能多的上清液尽可能到装有3毫升的L-15/10％FBS的培养基中在1％琼脂糖包被的培养皿，但是不干扰沉淀的心在离心管的底部。由于心的高达15％的可保留在上清液由于RNAlater中的高粘度，如在步骤3.7中所述检索它们。 </li><li>在化学罩，加0.5毫升的Trizol含有沉淀的心管和保持在冰上。 </li><li>根据荧光microscoPE，传送从1％琼脂糖包被培养皿含有的RNAlater / L-15/10％FBS的溶液的心（来自步骤3.5）转换成另一种1％琼脂糖包被培养皿用3ml的L-15/10％FBS稀释出RNAlater中。收集的心在培养皿的中心，下化学罩在小体积转移成沉淀心中的Trizol的管。 </li><li>涡1.5 ml管中含有的Trizol心中破坏心脏和孵化5分钟，在室温下。 </li><li>在化学罩，加100微升氯仿（警告），调匀（使用前以最大速度离心30秒）的Trizol /氯仿溶液转移到1.5 ml的预纺PLG管。孵育3分钟，在室温下进行。 注意:请阅读氯仿MSDS方可使用。处理氯仿试剂罩下，并建议穿戴个人防护装备。避免与皮肤，眼睛和衣服接触。远离不相容，如金属，碱。 </li><li> CentrifuGE将样品在15700×g离心15分钟，在4℃下，将水相转移到一个新的1.5毫升管。 </li><li>添加5-10微克糖原和250μl的预冷（-20℃）异丙醇;拌匀，孵育过夜，在-20℃。 </li><li>离心样品在15700×g离心30分钟，在4℃，弃去上清液。 </li><li>在15700×g离心在4℃下洗用1ml 75％乙醇，离心沉淀15分钟，并弃去上清液。 </li><li>让乙醇蒸发干燥的粒料在室温下，直到它变成白色（ 例如 10-15分钟）。 </li><li>添加20微升不含RNA酶的DDH 2 O的以沉淀孵育10-15分钟，在55℃以溶解的RNA然后保持在冰上。 </li><li>评估RNA的浓度和纯度通过吸光度测定。通过运行在1％琼脂糖凝胶的样品评估RNA的完整性。 </li></ol>

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The authors have nothing to disclose.

该协议允许斑马鱼胚胎心脏组织的快速富集的基因表达分析。心脏特异性RNA样品的数量和质量在很大程度上取决于几个关键步骤:第一，如果能够防止在该协议中的每一步的心损耗的样品的量有很大的提高，因为RNA纯化将仅具有足够的工作起始材料。第二，样品，这完全取决于实验者的纯度，通过分类和收集的心内的培养皿同时严格排除其它组织确定的。第三，样品的质量主要取决于限制可能发生...

斑马鱼（ 斑马鱼 ）被广泛用于发育生物学研究器官体内 ，由于其快速的，透明的和宫外胚胎发育，结合小尺寸和转基因记者线荧光蛋白的组织特异性表达的可用性。这个小脊椎动物特别适于研究心脏发育，因为早期的斑马鱼胚胎的氧合不依赖于心脏的跳动及血液流动;这些特点使大量心血管突变1,2的表征和斑马鱼现在是一个被广泛认可的模式生物来研究心脏疾病3。 到胚胎发育过程中研究基因表达，转录通常通过整个安装原位杂交（WISH）4分析，RT-qPCR的5，微阵列6，或下一代测序（RNA测序）7。而WISH允许基因表达的整个胚胎内的空间 - 时间分析，转录水平通过RT-qPCR的，微阵列或RNA测序方法，通常进行评估。然而，这些方法需要组织富集为特定的基因表达图谱。 因为斑马鱼胚胎心脏表示整个胚胎的一小部分，心脏发育过程中转录研究需要一个协议，用于解剖和心脏的富集。此外，为了得到生理上相关的数据，以维持心脏组织完全功能，直到RNA提取它是很重要的。在这里，我们描述了一种协议，用于快速分离生理正常和跳动心脏从数百斑马鱼胚胎的有效地获得高品质的RNA样品用于进一步分析。本方法是基于报道的烧伤和麦克雷，2006年8所述的协议，对于心脏组织的富集，这两种方法都使用的转基因心肌记者线并利用斑马鱼胚胎心脏与其他组织的鉴别粘附性能优势。简要地说，通过一个狭窄的枪头吹打许多胚胎上下，心脏被同时从胚体释放并随后从胚胎碎片两个快速过滤步骤中分离;荧光标记的心脏，然后手动地从剩余的碎片来分类并收集用于进一步处理。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr >
			<td >Equipment for raising fish and collecting eggs&#160;</td>
			<td></td>
			<td></td>
			<td>see the Zebrafish Book11 for details</td>
		</tr>
		<tr >
			<td >Fluorescence stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Refrigerated Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >UV-Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>eg. Thermo Scientific Nanodrop 2000</td>
		</tr>
		<tr >
			<td >Nucleic acid electrophoresis chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dishes 4cm &#216;, coated with 1% agarose in E3 medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipettes and tips (P20, P100, P1000)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1,5ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >15ml and 50ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Pair of Dumont #5 forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >ExactaCruz&#8482; Round Gel Loading Tips in Sterile Rack, 1-200&#956;l&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-201732</td>
			<td></td>
		</tr>
		<tr >
			<td >100 &#956;m filter (BD Falcon 100 mm Cell Strainer)</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr >
			<td >30 &#956;m filter (Pre-Separation Filters-30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >Phase lock gel, heavy, 1,5ml tubes&#160;</td>
			<td>Prime</td>
			<td>2302810</td>
			<td></td>
		</tr>
		<tr >
			<td >Egg water medium</td>
			<td></td>
			<td></td>
			<td>60&#956;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td>
		</tr>
		<tr >
			<td >E3 medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr >
			<td >Tricaine (3-amino benzoic acidethylester)</td>
			<td>Sigma-Aldrich</td>
			<td>A-5040</td>
			<td>4mg/ml Tricaine stock solution, pH 7</td>
		</tr>
		<tr >
			<td >1% agarose (in E3 medium)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1% agarose gel (in TBE buffer)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#180;s L-15 medium&#160;</td>
			<td>Gibco</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAlater</td>
			<td>Ambion</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizol</td>
			<td>Ambion</td>
			<td>1559606</td>
			<td></td>
		</tr>
		<tr >
			<td >Glycogen&#160;</td>
			<td>Invitrogen</td>
			<td>10814-010</td>
			<td>20 &#181;g/&#181;L in RNase-free water</td>
		</tr>
		<tr >
			<td >chloroform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >isopropanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >75% ethanol (in DEPC-ddH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nuclease-free water or sterilized DEPC treated ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nucleic acid loading buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >TBE (Tris/Borate/EDTA) buffer for electrophoresis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

斑马鱼胚胎的心脏是由只有几百细胞，占整个胚胎中只有一小部分。因此，为了防止被掩蔽由全局胚胎转录心脏转录，有必要收集足够数量的心作进一步的分析。此外，如斑马鱼心脏发育迅速进行，心脏收集和RNA提取方法需要快，以保证样品的均匀性。在这里，我们提出了一个快速的手动清扫协议收集功能/跳动的心，从斑马鱼胚胎。这是为随后的心脏特异性RNA提取，以确定心肌特异性基因表达水平通过转录组分析，例如定量实时聚合酶链反应（RT-qPCR的）的必要先决条件。该方法是基于在斑马鱼胚胎心脏和其他组织相比的差的粘合性能;这允许迅速的phy从心外心脏组织由流体剪切力的破坏，逐步过滤和人工采集转基因荧光标记心中的组合SICAL分离。

在这里，我们描述了使用斑马鱼的Tg（MYL7:GFP）的有代表性的心脏解剖实验专在心肌（图1）的转基因twu34线9，它表达绿色荧光蛋白（GFP）。我们收集了两个解剖心，从他们得出评估心脏样品的纯度胚胎。简言之，将纯合的Tg（MYL7:GFP）twu34斑马鱼9进行异型杂交用野生型，使得所有的胚胎心脏的GFP标记。大约500胚胎（56 HPF）转移到1.5毫升管（约100?...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

到斑马鱼心脏发育过程中分析的心脏基因表达分布，总RNA已被提取从离体心脏。在这里，我们提出了一个协议，用于收集功能/跳动的心迅速人工清扫，从斑马鱼胚胎获得心脏特异性mRNA。

大规模的斑马鱼胚胎的心脏解剖的转录分析

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

large-scale-zebrafish-embryonic-heart-dissection-for-transcriptional

Research

JoVE Journal

Developmental Biology

14.3K 次观看.  Max Delbrück Center for Molecular Medicine. 到斑马鱼心脏发育过程中分析的心脏基因表达分布，总RNA已被提取从离体心脏。在这里，我们提出了一个协议，用于收集功能/跳动的心迅速人工清扫，从斑马鱼胚胎获得心脏特异性mRNA。 

视频: Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...