The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

在动物身上进行的所有实验均委员会实验室动物护理和墨西哥自治国立大学（UNAM）使用批准的，由CICUAL协议号:FLC40-14。 （CICUAL:"科米特Institucional第下午CuidadoŸUSO德洛斯阿尼马莱斯德的Laboratorio德尔研究所德FisiologìaCelular，大学墨西哥国立自治"）。 1.胚胎蛋白提取物的制备<ol><li>收集100 - 200的胚胎为每个条件在所希望的阶段比较（morphants与野生型），例如72小时后受精（HPF）。 </li><li>在含鱼水之培养皿的地方胚胎（ 见表1）和手动dechorionate用细尖镊子胚胎。避免在这一步骤（或整个协议的任何其他蛋白酶），链霉蛋白酶，因为它可能消化针对性的受体。 </li><li>胚胎放置在1.5 ml离心管，洗胚胎TW用1×磷酸盐缓冲液（PBS）的冰（ 见表1）。 </li><li>加入500微升deyolk缓冲液（ 见表1）。 </li><li> （ - 40倍30）在溶液中的胚胎轻轻吹打向上和向下释放最蛋黄。 72高倍视野48 HPF胚胎使用蓝色和黄色的枪头，分别为。黄色提示可能需要稍微切割，以避免破坏胚胎。成功蛋黄释放可以通过显微镜下观察胚胎进行检查。 </li><li>离心600 XG 15秒收集胚胎。 </li><li>用吸管小心弃上清。 </li><li>洗涤胚胎两次用500μl洗涤缓冲液通过以最低速度轻轻涡旋（ 见表1）。 </li><li>离心600 XG 15秒收集胚胎。 </li><li>从这里出发，继续在4℃的过程。 </li><li>用吸管小心弃上清。 </li><li>重悬的胚胎在350微升裂解缓冲液（见表1），并使用塑料杵均化。 </li><li>孵育裂解胚胎搅拌30分钟上的试管摇杆在4℃。 </li><li>离心机以除去不溶的碎片裂解胚胎在11000×g离心15分钟。 </li><li>转印用吸管至新管清除上清液。 </li><li>确定由Bradford蛋白测定4，或其它合适的过程总蛋白。如果使用Bradford测定法，在洗涤剂礼物的当量浓度的裂解缓冲液的存在下进行校准曲线，因为它们会导致标准4的蛋白的低估。 </li></ol> 2.内源受体蛋白质的检测<ol><li> 亲和标记和免疫（所有这些步骤在4℃） <ol><li>放置400 - 500微克总胚胎蛋白质在1.5ml微量离心管中并稀释至1微克/微升用缓冲液1（ 见表1）。 注意:为了获得500微克总胚胎蛋白，大约100 - 200胚胎必须首先处理。 72 HPF胚胎常规收率总胚胎蛋白质，这是足够的β聚糖检测的〜5微克。 </li><li>加标记的TGF-β2的库存的足够体积，以达到150微米的最终浓度，并于4℃在试管摇杆2小时在搅拌孵育。 TGF-β2必须AFLIP开始前由氯胺T方法如通过Cheifetz 等人 5所述进行标记，用125 I。 注意:使用屏蔽，同时处理125 I标记配体以尽量减少暴露。 </li><li>添加针对感兴趣的受体未稀释的抗体的足够体积，以达到1:100稀释并在4℃下继续温育另外2小时（温育可以是O / N）。这是抗血清＃31中的最佳稀释度，在此研究中使用的和其他地方描述的3，但根据不同的一个的质量tibody，更大或更小的稀释可能是最佳的。 </li><li>加入50μl的合适的免疫球蛋白结合珠（例如，G蛋白 - 琼脂糖，这是以前在TNTE平衡并重新悬浮1:5在TNTE其原始体积的），并在4℃孵育50分钟以上的试管摇杆搅动C。 </li><li>恢复由微量离心珠粒在11000×g离心20秒。 </li><li>在适当的放射性垃圾容器弃上清。 </li><li>用1ml的IP洗涤缓冲液（ 见表1）的，通过涡旋和微量离心洗IP的珠粒三次在11000×g离心，每次20秒。 </li><li>悬浮IP-珠250微升IP洗涤缓冲。 </li><li>添加1.5微升二琥珀酰亚胺辛二酸酯的（DSS，溶解在DMSO中，在10毫克/毫升）。只是在这一步使用前准备DSS解决方案。 </li><li>在4℃搅拌下孵育15分钟。 </li><li>为了解渴交联反应，加入500μlØ˚FIP洗涤缓冲液，辅以足三Cl pH为7.4的股票达到25毫米的Tris-CL。 TRIS中游离氨基捕捉未反应的决策支持系统。 </li><li>离心机在11000×g离心20秒收集IP的珠并丢弃上清液。 </li><li>悬浮IP-珠在减少Laemmli缓冲液的30微升。 </li><li>煮沸样品在94℃5分钟。 </li><li>任选地，分析使用制造商的方案在γ计数器中的样品。 </li></ol></li><li> 样品分析 <ol><li>受试者样品变性SDS-PAGE。在对受体的质量的适当百分比使用聚丙烯酰胺和下标准程序运行凝胶。 </li><li>浸泡在固定液凝胶（ 见表1）在RT下30分钟。 </li><li>用蒸馏水冲洗凝胶15分钟。 </li><li>将凝胶在以前水合过滤纸和保鲜膜薄膜覆盖。 </li><li>在80℃进行1小时干凝胶。 </li><li>暴露在白色磷光SCRE凝胶恩在室温O / N。 </li><li>使用扫描使用制造商的协议的磷光暴露屏幕。 </li></ol></li></ol>

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The authors have nothing to disclose.

Western印迹用针对感兴趣的蛋白质的特异性抗体的使用是一种有价值的工具胚胎发育过程中以研究其表达7。然而，高度糖基化的蛋白质的免疫印迹尚未十分成功，由于其低效率的转让和弱结合到硝酸纤维素或PVDF膜8,9。 蛋白多糖是因为他们的带负电荷的，并且不很好地结合要么聚苯乙烯表面或疏水印迹膜共价结合的糖胺聚糖链（GAG）的这个缺点的一个很好的例子。此外，GAG链的大小异质性"传播"的蛋白质在凝胶的部门，每印迹的面积有效地减少其数量。此外，如果所关注的蛋白质具有表达水平较低，其检测由正规蛋白质印迹是一个困难的任务。 β受体（TGFBR3）或β聚糖III型转化生长因子（BG），与在斑马鱼3哺乳动物1两GAG的依恋网站和一个膜受体，具有所有这些属性。虽然，为了克服这些障碍的协议已经设计，如检测通过抗生物素蛋白-生物素复合物（ABC）的系统10,11或带正电荷的膜12蛋白聚糖的免疫印迹，它们不能解决在相同的协议这两个问题。趁着BG的高亲和性的其天然配体TGFβ2和特异性抗体的可用性，AFLIP，结合亲和标记和免疫沉淀的优点的技术中，上述讨论的蛋白印迹分析检测的限制可能被克服。 与他们的放射性标记的配体与膜结合的受体的亲合标记是一个很好的利用工具，已经工具为几个重要的生长的鉴定和表征中培养细胞13-16因子受体。而在以往的亲和标记细胞单层进行共价在组织培养皿的配体标记，然后裂解，在AFLIP首先形成在胚胎裂解物，然后通过免疫沉淀纯化，最后，共价交联的配体 - 受体复合物。因为它的使用放射性标记的配体的，AFLIP是非常敏感的，在原则上可以适于其他生长因子或细胞因子受体的量的标记的配体和良好的抗体是可用的。 AFLIP的这种潜在的，将有助于这些低丰富，但生理有关的分子检测。 因为在亲和标记的交联工序中的固有变异性，AFLIP不能用来定量确定所测量的受体的水平。但是，如果有足够的并行控制进行的，它可以提供它们的表达的相对水平的相当准确的计量。作为AFLIP使用总蛋白提取物，它不restri反恐执行局到受体中的一个特定的细胞的位置，它揭示了在整个个人或器官中的表达，如果施加到解剖成人标本。最后，如​​果需要的话AFLIP可能与其它生物化学协议，如碳水化合物酶消化，进一步表征感兴趣的受体。 类似于常规亲和标记，强烈建议新鲜125 I标记的配体。定的125 I的半衰期短，该配体必须在最初2周内用新鲜的同位素标记后使用。虽然大多数协议的警告已在相应的步骤中提到的，特别提及应当给予仔细去除蛋黄的，它必须为完全和均匀越好。留下不均量蛋黄会导致在裂解物善意胚胎蛋白质的不正确定量。

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3. 
However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3's proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

<table border="0" cellpadding="0" cellspacing="0" width="670">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Disuccinimidyl suberate (DSS)</td>
			<td style="width:168px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21555</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protein G Sephraose 4 Fast Flow</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">17-0618-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Gel Dryer Model 583&#160;</td>
			<td style="width:168px;">BIO-RAD</td>
			<td style="width:119px;">1651745</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Typhoon 9400</td>
			<td style="width:168px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">63-0055-78</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cobra II Auto gamma counter</td>
			<td style="width:168px;">Packard</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Exposure Cassette</td>
			<td style="width:168px;">Molecular Dynamics</td>
			<td style="width:119px;">63-0035-44</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Na2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">K2HPO4</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">3246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH4O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C2H4O2</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">9508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CH2O</td>
			<td style="width:168px;">J.T. Baker</td>
			<td style="width:119px;">2106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">L4509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">ED</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">C3306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:168px;">Fisher Scientific</td>
			<td style="width:119px;">S233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:168px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Crystal Sea Marine Mix</td>
			<td style="width:168px;">Marine Enterprises International</td>
			<td style="width:119px;">http://www.meisalt.com/Crystal-Sea-Marinemix</td>
			<td></td>
		</tr>
	</tbody>
</table>

affinity labeling detection of endogenous receptors from zebrafish embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

图1示出了具有AFLIP获得的代表性结果。在泳道1信号来自于125 I配体共价连接到或斑马鱼β聚糖核心蛋白（BG核心，150 kDa的标记下面）或已经由糖胺聚糖的附件（GAG，涂抹处理，以它的蛋白聚糖的形式对BG芯范围从170 kDa的凝胶的顶部）。迁移的这个模式中，锋利的核心蛋白加上涂蛋白聚糖（由于GAG链的长度异质），是TGFBR3 2的特征。由于DSS不共价连接形成每一个配体-受体复合物，有免费的125出现在迁移前凝胶（125 I-TGF-β2）的I-配体。此游离配体与受体结合，和免疫沉淀期间仍然约束，但由于它不是共价连接，所述的SDS-PAGE过程期间分离。没有netheless，其信号的强度仍然关联到由标记的受体给出的。这可以通过比较泳道2和3.在48高倍视野的斑马鱼的胚胎可以理解表现出较低量的BG的超过72 HPF胚胎3，这可通过在GAG和BG芯的微弱信号，即对应的强度可以看出由出现在凝胶前端的游离配位体给定信号（泳道2）。鉴于抗大鼠抗体BG 6做的ZF BG交叉反应，抗体在AFLIP使用时没有给出信号，因此，作为阴性对照（泳道3）。类似的负面结果与反ZF BG抗体3的免疫前血清获得。 图2示出AFLIP如何可以用来衡量特定受体的表达水平在经受实验操作的胚胎，在这种情况下，β聚糖减少由于在吗啉jection 3。泳道1显示TGFBR3的在72 HPF野生型胚胎的水平，而泳道2示出了施用7纳克定向到ZF BG初级转录物的外显子2-内含子边界吗啉代的的效果。注意，与此吗啉得到的BG下调不受其误匹配的对照（泳道3）再现。这些结果证实，如3之前描述的本啉得到的表型是特异于TGFBR3的击倒。 <img alt="图1" src="/files/ftp_upload/54405/54405fig1.jpg" /> 在斑马鱼胚胎的BG的 图1 AFLIP检测 。从胚胎在72小时后的受精（HPF）总蛋白提取物（泳道1和3）和48 HPF（泳道2）进行125 I TGF-β2亲和标记，随后用IP用兔抗斑马鱼BG（泳道1和2，ZBG）或兔抗大鼠BG（泳道3，RBG）作为对照。在放射自显影BG可以不GAG（BG芯）被检测或作为糖胺聚糖改性BG（GAG）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54405/54405fig2.jpg" /> 图2. BG吗啉击倒由AFLIP检测。来自未处理的WT胚胎（lane1），具有特定的BG吗啉（泳道2）或错配对照（泳道3）显微注射的BG的125 I TGF-β2亲和标记。每百万（CPM）计数恢复免疫沉淀和交联步骤指示后， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54405/54405fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-w第i next.within页="总是"&gt; <tbody><tr><td> 缓冲 </td><td> 组成 </td></tr><tr><td>缓冲区1 </td><td>的50mM的Tris-HCl pH为7.5，150 mM氯化钠，0.1％的Triton X-100 </td></tr><tr><td> Deyolk缓冲</td><td> 55 mM氯化钠，1.8毫摩尔的KCl，1.25毫碳酸氢钠 </td></tr><tr><td>鱼水</td><td>见材料表</td></tr><tr><td>固定液</td><td> 50％CH 3 OH，12％CH 3 COOH，0.185％甲醛</td></tr><tr><td> IP洗涤缓冲液</td><td>的10mM 的 Na 2 HPO 4，2mM的KH 2 PO 4，137 mM氯化钠，2.7 mM的氯化钾，0.1％的Triton X-100，0.02％SDS，pH 7.4的</td></tr><tr><td> Laemmli缓冲液</td><td> 2％SDS，10％甘油，100毫摩尔DTT，60毫摩尔Tris（pH值6.8），0.001％溴酚蓝。 </td></tr><tr><td>裂解缓冲液</td><td>的50mM的Tris-HCl pH7.4的，150毫摩尔NaCl，1mM EDTA中，1毫PMSF，0.5％的Triton X-100，0.1％SDS，0.5％Deoxych钠酚盐</td></tr><tr><td> PBS </td><td> 137 mM氯化钠，2.7 mM的氯化钾，10毫的 Na 2 HPO 4，2mM的KH 2 PO 4，pH7.4的</td></tr><tr><td> TNTE </td><td>的50mM的Tris-HCl pH7.4的，150毫摩尔NaCl，1mM EDTA中，0.1％的Triton X-100 </td></tr><tr><td>洗涤缓冲液</td><td> 110 mM氯化钠，3.5毫摩尔的KCl，2.7毫摩尔氯化钙 ，10mM的Tris-盐酸pH 8.5的</td></tr></tbody></table> 表1:缓冲组合物

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

从斑马鱼胚胎内源性受体亲和标记检测

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

5.6K 次观看.  Universidad Nacional Autónoma de México. A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

视频: Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

科研

发育生物学

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

DNA的特定染色甲基绿在胚胎的荧光标记的应用

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

斑马鱼胚胎的倍性操作与热休克治疗2

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

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 zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

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 ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

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. ...