这项研究是由MRC和BBSRC的补助金。我们感谢阿加塔Zieba和卡捷琳娜Pardalis乌尔夫Landegren的实验室，乌普萨拉大学设立的技术和有用的意见帮助。 Olink Biosience技术帮助和演示。

1。电镀细胞和治疗与BMP <ol><li>文化Neuro2a GMEM细胞与10％胎牛血清，1个非必需氨基酸（NEAM），1个丙酮酸钠和1x L -谷氨酰胺。 16井室幻灯片使用胰蛋白酶（0.05％EDTA）和15.000-20.000板，每孔细胞分裂的细胞。 辅以10％FBS，1个L -谷氨酰胺和1x青霉素/链霉素的DMEM文化HEK293T或COS7细胞。消毒浸泡在70％的乙醇和使用Immedge画笔画出1厘米2井polysine幻灯片。使用50μL培养基胰蛋白酶（0.05％EDTA）和20.000-25.000板，每孔细胞分裂的细胞。 </li><li>细胞在37 °彗星在饱和湿度，5％CO2培养箱。 </li><li> 24 h后更换为无血清GMEM（0.1％白蛋白，NEAM 1X，1X丙酮酸钠和1x L -谷氨酰胺的补充）Neuro2a，或无血清DMEM（L -谷氨酰胺和1x青霉素/链霉素补充）HEK293T培养基/ COS7，并培育2-3个小时的细胞保持细胞与正常培养条件下（即10％胎牛血清）作为对照组使用的三口井。 </li><li>治疗所需的时间与dorsomorphin（BMP通路，2微米抑制剂）或（25纳克/毫升）BMP4的细胞（如为10分钟，1 h）5。 </li></ol> 2。固定和通透<ol><li>准备固定液4％PFA。称取4克煤灰，准备在100毫升PBS的4％的解决方案。暖的解决方案，在50 ° C直到它是明确的。通过0.22微米的过滤器和储存在4 ° C过滤解决方案，直到使用。使用新鲜固定液（存储不超过2天）。 </li><li>从水井中吸用1xPBS。不要吸管直接到细胞中的解决方案，因为这可能会导致细胞脱离。下一步之前，如果使用玻片删除商会，但离开井周围的硅。 </li><li>加入50微升4％PFA和孵化为10分钟，在室温下，无搅拌。 </li><li>用PBS冲洗3 × 5分钟，在室温下搅拌一个Coplin JAR细胞。 </li><li> 0.5％的Triton X - 100的PBS在室温下搅拌10分钟，没有治疗的细胞。 </li><li> 0.05％吐温20的TBS（TBS - T）洗涤3 × 5分钟，在室温下搅拌一个Coplin JAR细胞。 </li></ol> 3。封锁<ol><li>点击关闭TBS - T。添加一滴Duolink第二封闭液（1X），每孔。 </li><li>孵育的幻灯片，在1小时预热湿度室，在37 ° C。 </li></ol> 4。一抗<ol><li>混合，并在1:100稀释aP-Smad1/5/8（多克隆）和Smad4的1:100（鼠单克隆）Duolink第二抗体稀释液（1X）。也aP-Smad1/5/8准备和A - Smad4的单独控制使用在相同浓度。 </li><li>挖掘从幻灯片封闭液。删除尽可能多的阻塞解决方案，但要特别小心，不要让之前加入抗体干细胞。添加40μL的抗体解决方案水井。在一个只添加额外的阴性对照抗体稀释液。 </li></ol> 5。解放军探头<ol><li>淡化两个解放军探头在抗体稀释液（Duolink II抗鼠减号和Duolink二抗兔游）1:5。 </li><li>挖掘从幻灯片的主要抗体溶液。洗2次5分钟的幻灯片每个1X Duolink II洗涤缓冲液中的一个在室温下搅拌Coplin的JAR。 </li><li>塔从幻灯片的洗涤缓冲液和添加解放军探头解决方案（40​​μL/孔）。 孵育的幻灯片，在1小时预热湿度室，在37 ° C。 </li></ol> 6。结扎<ol><li>旋涡Duolink第二结扎股票（5倍）和高纯度水和混合稀释1:5。计算水量时，考虑的一个最终稀释度为1:40，将在加入之前，除了混合井连接酶的量。 </li><li>挖掘解放军从幻灯片探头解决方案。在1x洗涤缓冲液清洗2次5分钟，每一个Coplin JAR在室温下搅拌幻灯片。 </li><li>从使用冻结块的冷冻室（-20℃），取出连接酶。添加连接酶结扎解决方案，在1时40的稀释和旋涡（6.1准备）。 </li><li>塔从幻灯片的洗涤缓冲液和结扎连接酶的解决方案添加到每口井（40μL/孔）。孵育30分钟的预热湿度室，在37 ° C。幻灯片</li></ol> 7。放大注:光敏感的试剂。保持避光的幻灯片。 <ol><li> Duolink第二扩增股票（5倍）1:5的高纯度水和混合稀释。当计算水量考虑到之前的组合除了日将在最后的1:80稀释添加聚合酶量Ë井。 </li><li>挖掘从幻灯片结扎连接酶解决方案。在1x洗涤缓冲液清洗幻灯片与为2分2次在每一个Coplin JAR在室温下搅拌。 </li><li>从使用冻结块的冷冻室（-20℃）聚合酶。聚合酶扩增解决方案（7.1准备）在1:80的稀释和涡。 </li><li>点击关闭从幻灯片的洗涤缓冲液，并添加扩增聚合酶的解决方案，以每口井（40μL/孔）。在100分钟的预热湿度室，在37 ° C。孵育幻灯片</li></ol> 8。成像的制备注:光敏感的试剂。保持避光的幻灯片。 <ol><li>点击幻灯片扩增聚合酶的解决方案，并在Coplin JAR洗2次10分钟每个1X Duolink第二洗涤缓冲液B在室温下搅拌。 </li><li>浸在0.1 ×洗涤缓冲液B.幻灯片</li><li>完全从16以及幻灯片取出硅胶。添加〜40μlDuolink第二安装用DAPI盖玻片中等，并轻轻地按下它稍微使有盖玻片下无气泡的样本，的地方。修复和密封的盖玻片上使用指甲油的幻灯片。至少15分钟前等待出发，成像。 </li><li>共聚焦荧光显微镜分析样品。获得数字图像。 </li></ol> 9。量化注:光敏感的试剂。保持避光的幻灯片。 <ol><li>使用Duolink ImageTool依靠图像的信号，以获得测量信号级别。 </li><li>比较测量，并作出图形。 </li></ol> 10。代表性的成果 原位解放军实验结果表明:作为离散的荧光斑点。信号的位置取决于特定蛋白质的研究。 <img alt="图1" src="/files/ftp_upload/2631/2631fig1.jpg" /> 图1 原位解放军。BMP4的刺激后的Neuro2a细胞。 2微米dorsomorphin（BMP的途径抑制剂）（A）或25毫微克/毫升BMP4的细胞用（B）为60分钟或不及时治疗（CE认证）。对P-Smad1/5/8和Smad4抗体被用来探测积极配合物A和B的主要抗体aP-Smad1/5/8单（三）和Smad4的单独（四）或遗漏的主抗体（五）作为对照。蓝色:DAPI;红:解放军信号。在Leica SP5的共聚焦显微镜照片获得。 （六）解放军信号Duolink ImageTool软件计算，平均每个细胞的细胞核中的斑点是在图。 （七）Neuro2a细胞离开Dorsomorphin（2微米）或60分钟（25毫微克/毫升）BMP4的未经处理或处理。细胞裂解和蛋白SDS - PAGE和分析aP-Smad1/5/8，增殖细胞核抗原免疫加载控制。 （*）非特异性带。目前在复杂与Smad4的注意，aP-Smad1/5/8抗体无法区分3种不同的蛋白质。

<ol>
	<li>Miyazono, K., Kamiya, Y., Morikawa, M. B. o. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=morphogenetic+protein+receptors+and+signal+transduction.">morphogenetic protein receptors and signal transduction.</a> J Biochem. 147, 35-51 (2010).</li><li>Gustafsdottir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proximity+ligation+assays+for+sensitive+and+specific+protein+analyses.">Proximity ligation assays for sensitive and specific protein analyses.</a> Anal Biochem. 345, 2-9 (2005).</li><li>Soderberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+individual+endogenous+protein+complexes+in+situ+by+proximity+ligation.">Direct observation of individual endogenous protein complexes in situ by proximity ligation.</a> Nat Methods. 3, 995-1000 (2006).</li><li>Soderberg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+proteins+and+their+interactions+in+cells+and+tissues+using+the+in+situ+proximity+ligation+assay.">Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.</a> Methods. 45, 227-232 (2008).</li><li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism.">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nat Chem Biol. 4, 33-41 (2008).</li><li>Baan, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+proximity+ligation+detection+of+c-Jun/AP-1+dimers+reveals+increased+levels+of+c-Jun/Fra1+complexes+in+aggressive+breast+cancer+cell+lines+in+vitro+and+in+vivo.">In situ proximity ligation detection of c-Jun/AP-1 dimers reveals increased levels of c-Jun/Fra1 complexes in aggressive breast cancer cell lines in vitro and in vivo.</a> Mol Cell Proteomics. 9, 1982-1990 .</li><li>Massinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interaction+of+DYX1C1+with+estrogen+receptors+suggests+involvement+of+hormonal+pathways+in+dyslexia.">Functional interaction of DYX1C1 with estrogen receptors suggests involvement of hormonal pathways in dyslexia.</a> Hum Mol Genet. 18, 2802-2812 (2009).</li><li>Thymiakou, E., Zannis, V. I., Kardassis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+functional+interactions+between+liver+X+receptor/retinoid+X+receptor+and+Sp1+modulate+the+transcriptional+induction+of+the+human+ATP+binding+cassette+transporter+A1+gene+by+oxysterols+and+retinoids.">Physical and functional interactions between liver X receptor/retinoid X receptor and Sp1 modulate the transcriptional induction of the human ATP binding cassette transporter A1 gene by oxysterols and retinoids.</a> Biochemistry. 46, 11473-11483 (2007).</li></ol>

生产这种视频文章是由OLINK赞助人在接近结扎检测过程中使用的试剂。

原位可视化的蛋白复合物的需求量很大，特别是在信令蛋白质相互作用和蛋白质修饰细胞发出一个信号，从他们的表面到细胞核中使用的手段的研究。它已不可能，可视化和量化原位免疫前的两个内源性蛋白复合物。抗体共定位展品分辨率低和不能用于可视化真实的相互作用。 PLA是一种新技术，研究人员已经开始在不同的系统使用了巨大的成功6，7。在这里，我们展示了如何使用解放军不仅形象化，还要量化之间的内源性激活BMP的刺激效应随着时间的推移下游的Smad复合物。我们使用了不同的组织细胞培养，包括Neuro2a和依赖于在不同的物种（鼠标Smad4和兔aP-Smad1/5/8）提出了实现这一目标（图1）商业抗体。这种方法使我们首次看到随着时间的推移BMP的效应复合体的激活，而不是只是存在磷酸化Smad1/5/8，这可能不是所有的从事积极配合物与Smad4的。我们计算的信号（图1F），并从相同的细胞（图1G）免疫印迹获得量化的测量相比。我们得出的结论是斑点的数量提供了一个准确的信号随着时间的推移水平比较测量。该技术也适用于其他细胞系（HEK293T和COS7）与类似的结果（数据未显示）。 该技术的原理是基于与Duolink试剂盒提供了两个独特的探头。每个解放军探头由次要抗体连接到一种独特的合成寡核苷酸，作为一个记者的行为。探头附近允许的确切位置，这些探测器在接近连接DNA结扎。的寡核苷酸的距离，这使得DNA杂交和结扎小（&lt;40nm工艺），因此，交互的唯一的蛋白质可以使结扎。然后可以结扎的DNA扩增和扩增序列标记的寡核苷酸杂交检测。放大特定的，因为它取决于DNA - DNA杂交原则，还提供了高灵敏度的结扎的DNA被放大数倍，导致提高初始结扎事件。扩增的DNA杂交检测标记的寡核苷酸，并产生显着的，相当大的地方。由于可以算作点，解放军信号提供不仅精确的空间信息（交互事件的位置），但也一个客观量化这些事件2-4的方式。 可用于检测蛋白质相互作用的其他技术，包括共沉淀，免疫荧光和荧光共振能量转移（FRET）。免疫共沉淀分析被广泛使用使蛋白质复合物的隔离。该方法是伴随着免疫，这意味着必须在相对较高的水平表达，蛋白质，才能被检测到。高背景或某些蛋白质的非特异性结合珠问题有时难以克服的免疫共沉淀。此外，免疫共沉淀检测允许检测的蛋白质，可以是一种蛋白质复合体的一部分，但不能区分那些非常接近物理相互交融的接近。此外，合作immunoprecipitations不能提供定量分析，在单细胞分辨率或在细胞内的蛋白质复合物的本地化信息。在一个细胞或一个细胞通过免疫荧光舱蛋白质复合物并存的检测是基于散射信号的重叠，因此​​，具有较低的空间分辨率和蛋白质相互作用不能提供准确的信息。免疫荧光信号的量化，可以通过目测，只有当不同的是数倍，并不能准确或解放军点衡量。 FRET技术，允许活细胞中的蛋白质复合物的可视化和克服抗体和暴露的抗原表位的需要。然而，FRET技术通常取决于人工融合蛋白，这可能不是反映解放军的内源性蛋白复合物中的表达。 解放军协议中的阻塞条件是至关重要的，因为抗体聚集可能会出现增加的背景。抗体产品表中可以找到替代阻止条件。小学和中学的抗体遗漏适当的控制提供了可能有什么不妥的信息，他们必须始终包含。屏幕不同的抗体，这将使它们是单独使用时至少背景和最佳的信号，当他们在一起是很重要的。固定CELLS是至关重要的过度操纵可能导致蛋白质复合物，以充分变性和降解，而固定不足可能导致在过程中的步骤，以蛋白质复合物的损失。这是可以控制的固定液的浓度降低到3％，或限制在固定的时间。然而，固定还取决于是否主要抗体识别变性的蛋白或不。由于解放军的技术非常敏感，需要特别小心保持的孵育时间和不同样品之间的平等的微观条件。 样品前的最后一步在任何情况下不应该留下来干。干燥的样品，可以导致增加的背景。封闭液彻底清除也很关键，以避免背景。洗缓冲区应始终使用前室温，低温减缓酶促反应。对于进一步的故障排除参考手册的工具包。 这是很有用的污渍确定解放军信号（例如DAPI，肌动蛋白等）的亚细胞定位。在这里，我们使用的DAPI（安装介质中）细胞核染色。解放军是兼容的免疫抗体来确定类型的细胞或细胞区室（此处未显示）。可以做到准确的定量计数信号，并利用特定的软件（Duolink图像工具，Olink Biosience）。 总之，我们已经演示了如何使用解放军在原地，使用Duolink试剂盒检测BMP信号在固定细胞，并介绍其优点:易于使用，高度敏感的，没有任何背景，和一种独特的能力量化积极BMP 信号在原地。 运用这种技术来研究不同的蛋白质相互作用的限制取决于承认正在调查中蛋白质的主要抗体的可用性和质量。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>GMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21710025</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21063029</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>AutogenBioclear</td>
<td>7.01</td>
<td>&nbsp;</td>
<tr>
<td>Non-essential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11149068</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Pyruvate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11360070</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin / Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140122</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030-054</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300-054</td>
<td>&nbsp;</td>
<tr>
<td>16-well chamber slides</td>
<td>&nbsp;</td>
<td>Lab-Tek</td>
<td>178599</td>
<td>&nbsp;</td>
<tr>
<td>Polysine</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>631-0107</td>
<td>&nbsp;</td>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>631-0138</td>
<td>&nbsp;</td>
<tr>
<td>a-pSmad1/5/8</td>
<td>&nbsp;</td>
<td>Cell Signaling</td>
<td>9511</td>
<td>&nbsp;</td>
<tr>
<td>a-Smad4 (B8) </td>
<td>&nbsp;</td>
<td>Santa Cruz Biotechnology</td>
<td>sc-7966</td>
<td>&nbsp;</td>
<tr>
<td>Dorsomorphin</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>171260-10MG</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant Mouse BMP-4</td>
<td>&nbsp;</td>
<td>R&amp;D</td>
<td>5020-BP-010</td>
<td>&nbsp;</td>
<tr>
<td>PFA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6148</td>
<td>&nbsp;</td>
<tr>
<td>Triton-X 100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>H5151</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II Detection Reagents (Orange)</a></td>
<td>&nbsp;</td>
<td>OLINK</td>
<td>92007-0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II PLA probe anti-Mouse MINUS</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>92004-0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II PLA probe anti-Rabbit PLUS</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>92002-0100</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II Mounting Medium with DAPI</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>82040-0005</td>
<td>&nbsp;</td>
<tr>
<td><a href="http://www.olink.webbhuset.com/products_services/duolink_products.php?menu=380">Duolink II Wash Buffers Fluorescence</a></td>
<td>&nbsp;</td>
<td>Olink Bioscience</td>
<td>82049</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

detection of signaling effector-complexes downstream of bmp4 using in situ pla, a proximity ligation assay

骨形成蛋白是负责广泛的发展和生物效应。 BMP受体激活（磷酸化），然后，形成与Smad4的一个复杂的，并转运到细胞核内转录因子的功能启动BMP具体的下游效应1 Smad1/5/8效应。传统的免疫荧光技术对磷酸化的Smad肽抗体表现出低灵敏度，高背景，并提供总值量化，因为他们依靠的抗体信号的强度，特别是如果这是光敏荧光。此外，磷酸化的Smads的可能不是所有与Smad4的复杂和从事活跃的转录。 解放军在原地 ，是一种技术，能够检测具有高特异性和灵敏度 2-4的蛋白质相互作用。这项新技术的夫妇扩增的D​​NA的蛋白质的替代品的抗体识别。它会产生一个局部的，离散信号揭示承认事件的确切位置点的形式。信号的数量可以计算和比较提供了一个测量。我们采用原位解放军，使用Duolink套件，与抗体结合，允许的BMP信号效应phospho-Smad1/5/8和Smad4的检测，只有当这些接近，即在一个复杂的，只发生与信号激活。这使得首次，内源性BMP的可视化和测量时间课程实验中BMP4的刺激下具有高特异性和灵敏度信号。

Watch this Scientific Journal Video about Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay at JoVE.com

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

在这里，我们展示了如何使用接近结扎含量（解放军），与抗体结合的可视化骨形态发生蛋白（BMP），在固定的细胞信号。这种技术允许我们遵循激活效应的复合物的内源性BMP的核积累和BMP4的刺激下，他们的水平随着时间的推移进行量化。

检测信号效应复合物BMP4的下游使用原位解放军，接近结扎分析

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

detection-signaling-effector-complexes-downstream-bmp4-using-situ-pla

Research

JoVE Journal

Biology

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

31.6K 次观看.  Imperial College, Hammersmith Hospital. 在这里，我们展示了如何使用接近结扎含量（解放军），与抗体结合的可视化骨形态发生蛋白（BMP），在固定的细胞信号。这种技术允许我们遵循激活效应的复合物的内源性BMP的核积累和BMP4的刺激下，他们的水平随着时间的推移进行量化。

视频: Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

科研

生物学

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

检测和DNA损伤分析小鼠骨骼肌<em&gt;原位</em&gt;使用TUNEL法

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), are crucial hubs for cellular signaling and cell fate. Particularly, these inter-organelle contact sites allow the transfer of calcium from the ER to mitochondria through the voltage-dependent anion channel (VDAC)/glucose-regulated protein 75 (GRP75)/inositol 1,4,5-triphosphate receptor (IP3R) calcium channeling complex. While this subcellular compartment is under intense investigation in both physiological and pathological conditions, no simple and sensitive method exists to quantify the endogenous amount of ER-mitochondria contact in cells. Similarly, MAMs are highly dynamic structures, and there is no suitable approach to follow modifications of ER-mitochondria interactions without protein overexpression. Here, we report an optimized protocol based on the use of an in situ proximity ligation assay to visualize and quantify endogenous ER-mitochondria interactions in fixed cells by using the close proximity between proteins of the outer mitochondrial membrane (VDAC1) and of the ER membrane (IP3R1) at the MAM interface. Similar in situ proximity ligation experiments can also be performed with the GRP75/IP3R1 and cyclophilin D/IP3R1 pairs of antibodies. This assay provides several advantages over other imaging procedures, as it is highly specific, sensitive, and suitable to multiple-condition testing. Therefore, the use of this in situ proximity ligation assay should be helpful to better understand the physiological regulations of ER-mitochondria interactions, as well as their role in pathological contexts.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

研究内质网和线粒体相互作用通过<em&gt;原位</em&gt;接近连接分析在固定细胞

Structural interactions between the endoplasmic reticular (ER) and mitochondrial membranes, in domains known as mitochondria-associated membranes (MAM), ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

时空映射和粒子分析技术在细胞内 ca2 +原位信令定量中的应用

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

使用酶联免疫吸附测定法检测按蚊中的疟原虫孢子

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female ...

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. Despite the clinical significance of scar formation and impaired wound healing, our understanding of fascia fibroblast dynamics in wound healing is cursory due to the lack of relevant assays that enable direct visualization of fibroblast choreography and dynamics in complex environments such as in skin wounds. This paper presents a protocol to generate ex- situ skin scars using SCAD or &#34;SCar-like tissue in A Dish&#34; that emulate the complex environment of skin wounds. In this assay, 2 mm full-thickness skin is excised and cultured upside down in media for 5 days, during which scars and skin contractures develop uniformly. This methodology, coupled with fibroblast-lineage specific transgenic mouse models, enables visualization of individual fibroblast lineages across the entire wound repair process. Overall, this protocol aids&#160;researchers in understanding fundamental processes and mechanisms of wound repair,&#160;directly exploring the effects of modulators on wound healing outcomes.

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay

This protocol describes the generation of a skin-fascia explant termed "SCar like tissue in A Dish" or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

使用SCAD测定可视化疤痕形成 - 一种 非原位 皮肤疤痕测定

The mammalian global response to sealing deep tissue wounds is through scar formation and tissue contraction, mediated by specialized fascia fibroblasts. ...

用于微生物群多组学的增强细菌细胞外囊泡分离

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

作者聚焦：加速细菌细胞外囊泡分离和异质性的研究 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.