我们感谢所有在我们的实验室谁促成了优化和验证协议的人。这项工作是由INSERM和国家研究机构（ANR-09-JCJC-0116和ANR-11-BSV1-033-02）的支持。 ET博士学位由高等教育和研究的法国文化部的一个研究奖学金期间的支持。

1.溶液的制备<ol><li>通过稀释5.5毫升37％甲醛的14.5毫升的PBS制备于PBS（低盐）10％的甲醛。制备1M甘氨酸，pH 8.0的，溶解在50ml的PBS将3.8g甘氨酸;稀释此溶液，得到在1×PBS中的100mM甘氨酸。 </li><li>制备0.1％的Triton-X100在1×PBS中。通过使用3.0M的氯化钠和0.30柠檬酸三钠在25℃在去离子水缓冲液中制备具有pH为7.0制备20倍盐水柠檬酸钠（SSC）。这稀释缓冲1X和使用去离子水0.01X。 </li></ol> 2.细胞固定 注意:我们使用在这项研究中的HuH7肝细胞癌细胞系，但这种方法也适用于其他的粘附细胞培养物。 <ol><li>板的HuH7细胞（在DMEM 1g / L的葡萄糖进行培养，补充有10％胎牛血清和0.01％青霉素 - 链霉素的库存）以每未涂覆35毫米玻璃底培养皿150,000个细胞。当原电池工作文化，用胶原蛋白涂层的菜肴。 </li><li>次日，除去培养基。清洗细胞用1毫升PBS和吸出的。通过加入1ml的甲醛10％固定细胞，并孵育在搅拌下室温（RT）10分钟。 </li><li>停止用1ml的1M甘氨酸使反应并通过旋转混合。除去停止反应溶液并通过加入1ml 1×PBS中洗细胞;通过旋转和吸搅动。 1毫升的100mM甘氨酸添加到细胞中，并孵育它们在室温下搅拌15分钟，然后吸出。 注:该协议可以在这里停了下来，下面的步骤可以推迟到另一天。在这种情况下，加入1ml的100mM甘氨酸和，保持在4℃，如果必要的。 </li></ol> 3.细胞透<ol><li>加入1ml 0.1％的Triton-X100的在1×PBS中，在室温下孵育在搅拌下15分钟，然后吸出。上一次电池文化性工作时20分钟 - 在该温育时间可以提高到15ES（ 如初级小鼠肝细胞）。加入1 ml 1X PBS洗细胞;吸出。 </li></ol> 4.封锁<ol><li>添加40微升的封闭溶液以每个样品（由试剂盒提供）;这个体积可以以覆盖样品增加。在湿度室中孵育30分钟的菜在37℃。 </li><li>点击过的菜封闭液。试图获得平等的残液量为每张幻灯片，因为这会影响可重复性。不要让样品干！ </li></ol> 5.一级抗体<ol><li>稀释在1×PBS中的一抗，并添加溶液的培养皿（VDAC1小鼠抗体:1/100，IP3R1兔抗体:1/500）。可替代地，GRP75或CypD抗体（在1/500同时使用小鼠抗体）可以用来代替VDAC1。 </li><li>在4℃的湿度室中孵育过夜。洗载玻片使用的Tris缓冲盐水两次含0.01％吐温（TBS-T）。 </li></ol><p类="jove_title"&gt; 6。接近结扎分析探针<ol><li>接近结扎分析探针由试剂盒。根据初级抗体的物种选择探针。 </li><li>在抗体稀释液5:准备两个接近结扎分析探针1。使混合物静置在室温20分钟。添加稀释接近连接测定探头的解决方案。孵育在预加热的湿度室中的菜肴1小时，在37℃。用TBS-T洗碗两次。 </li></ol> 7.结扎<ol><li>使用在原位检测试剂德克萨斯红套件。 </li><li>稀释5倍结扎股票（由试剂盒提供）1:5的高纯度水拌匀。稀释1倍连接液（由试剂盒提供）连接酶1:40和旋涡。等待添加连接酶，直到立即除了样品之前。 </li><li>添加该溶液至每个样品（40微升为35毫米的玻璃底菜）孵育玻片在预热的湿度湛误码率在37℃，30分钟。用TBS-T洗载玻片两次。 </li></ol> 8.放大注意:小心，光敏感的试剂。 <ol><li>稀释5倍放大股票（由试剂盒提供）1:5的高纯度水。使用冷冻块中删除从冷冻聚合酶（-20℃）。稀释聚合酶（试剂盒提供）1:80在1倍放大的解决方案和旋涡。立即用混合前添加聚合酶。 </li><li>添加该溶液至每个样品（40微升为35毫米的玻璃底菜）。孵育在预加热的湿度室100分钟的幻灯片在37℃。挖掘出幻灯片的扩增聚合酶溶液。 </li></ol> 9.最后经洗涤<ol><li>洗于1×SSC洗涤缓冲液将载玻片2分钟。洗在0.01X SSC洗涤缓冲液将载玻片2分钟。让菜在黑暗中在室温下干燥。 </li></ol> 10.准备成像<ol&gt; <li>使用含有DAPI（水相和不固化）安装介质的最小体积，以确保没有气泡得到盖玻片下抓装入滑动。指甲油可以用来密封边缘。 （:594纳米，发射:624纳米，放大倍数:63X激励）使用荧光或共聚焦显微镜分析前，等待约15分钟。 </li><li>成像后，存放载玻片在黑暗中在-20℃。 </li></ol>

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The authors declare that they have no competing financial interests.

Collectively, our studies indicate that the in situ proximity ligation assay is truly a relevant strategy to follow and quantify endogenous ER-mitochondria interactions in fixed cells, without the need for using organelle-specific fluorophores or fluorescent proteins. The specific use of VDAC1/IP3R1 antibodies has been adapted to study ER-mitochondria interactions in HuH7 cells. However, alternative isoforms of VDAC and IP3R may be used, depending on the cell type. In this case, antibodies need to be validated b...

线粒体和内质网（ER）是不是在细胞中独立的细胞器，但它们在结构上和功能上相互作用在定义为线粒体相关的内质网的膜（MAM）的接触位点。事实上，MAMS对应其中ER的膜和线粒体紧密并列的区域，使从两侧的蛋白质之间的相互作用。然而，这些细胞器的膜不这些区域内熔合，因此它们保持它们的独立的实体。该MAMS起到钙（Ca 2+）关键作用，磷脂转移从ER到线粒体，影响能量代谢和细胞存活1-3。 内质网和线粒体之间的关系在20世纪70年代用电子显微镜首次观察。自那时以来，透射电子显微镜4,5，电子断层扫描6,7或ER和特定线粒体-荧光团的免疫定位S /荧光蛋白8人经典用于研究ER-线粒体相互作用。为MAM的分析的另一个有用的工具是基于使用亚细胞分离的。它允许MAM馏分的隔离通过耦合到Percoll密度梯度9差动超速离心。然而，最终产物含有丰富的MAM馏分，而不是纯的级分。总之，这些策略没有特别敏感和/或定量的，它们是不容易适合于大的筛选。可替代地，使用药物诱导的荧光间细胞器的接头遗传方法已经出现，但它们不允许细胞器相互作用的分析在蛋白10的内源表达水平。 根据Szabadkai的发现的IP3受体/ GRP75 / VDAC络合物在MAM 11中 ，我们开发了一个定量的方法来分析ER-线粒体相互作用。我们使用原位接近ligati上测定来检测和量化VDAC1和IP3R1之间的相互作用，在固定单元12参与在MAM接口的钙 -channeling复杂二细胞器表面蛋白。简言之，我们在线粒体外膜（小鼠抗VDAC1初级抗体）与IP3R1在ER膜（兔抗IP3R1初级抗体）（图1，分图a）探测VDAC1。然后，根据该测定，我们增加既抗小鼠和抗兔IgG（小鼠和兔接近结扎分析探针），其偶联至互补寡核苷酸的扩展。如果两个目标蛋白质是在低于40纳米的距离，寡核苷酸可以与随后加入的接头的寡核苷酸杂交以允许环状DNA模板（图1，图b）的形成。此环形DNA分子连接并放大，产生共价连接到邻近探针之一的单链DNA产物（图1，分图c） </stroNG&gt;。由于在MAM接口的ER和线粒体之间的距离为10纳米的范围内，以25纳米6，接近结扎和扩增可以完成，从而导致后续检测由于德克萨斯红标记的寡核苷酸探针（图1，图d的杂交）。每个荧光点代表VDAC1 / IP3R1之间的相互作用，因而允许在单个细胞中原位 ER-线粒体相互作用的定量。 <img alt="图1" src="/files/ftp_upload/54899/54899fig1.jpg" /> 图1:由内质网线粒体相互作用检测的示意图原位接近结扎分析。 一）针对VDAC1和针对IP3R1可以在MAM接口绑定到其表位接近兔第一抗体的小鼠初级抗体， 二）除了一对接近结扎探针的针对小鼠和兔IgG。这些探针已附加的DNA链，可以形成用于连接寡核苷酸的结扎模板。 三）结扎后所形成的环状DNA链可以通过使用德克萨斯红标记的寡核苷酸扩增和d）通过显微镜观察作为荧光点。 <a href="http://ecsource.jove.com/files/ftp_upload/54899/54899fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 原位接近连接测定实验相似可以与GRP75 / IP3R1对抗体，以及亲环D（CypD）来执行/ IP3R1抗体，考虑到CypD被证明与在MAM接口的IP3受体/ GRP75 / VDAC复合物相互作用12-14。

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Formaldehyde</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">F-8775</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Glycine</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">G-8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Triton</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">T8532</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">35mm Glass bottom culture dishes</td>
			<td style="width:169px;">MatTeK corporation</td>
			<td style="width:187px;">P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Blocking solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92004 or DUO-92002</td>
			<td>provided in the Duolink PLA probes, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">VDAC1 antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:187px;">ab14734</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">IP3R1-H80 antibody</td>
			<td style="width:169px;">Santa Cruz</td>
			<td style="width:187px;">sc28614</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CypD antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:187px;">ab110324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Grp75 antibody</td>
			<td style="width:169px;">Santa Cruz</td>
			<td style="width:187px;">sc13967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">TBS 10X</td>
			<td style="width:169px;">euromedex</td>
			<td style="width:187px;">ET220</td>
			<td>Dilute to obtain 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Tween 100X</td>
			<td style="width:169px;">euromedex</td>
			<td style="width:187px;">2001-B</td>
			<td>dilute in TBS to obtain 0,01%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PLA Probes Mouse MINUS</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92004</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PLA Probes Rabbit PLUS</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92002</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Duolink detection reagents red</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ligation solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Ligase</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Amplification solution</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Polymerase</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO-92008</td>
			<td>Part of the Duolink detection reagents red, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Duolink Mounting Medium</td>
			<td style="width:169px;">Sigma</td>
			<td style="width:187px;">DUO80102</td>
			<td>Duolink, Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Softwares:</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Blob-finder software</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td>BlobFinder is a freely distributed software that can perform calculations on cells from fluorescence microscopy images. This software can be downloaded for free from The Centre for Image Analysis&#160;at Uppsala University who have developed the software and the work was supported by the EU FP6 Project&#160;ENLIGHT&#160;and&#160;&#160;Olink Bioscience. http://www.cb.uu.se/~amin/BlobFinder/index_files/Page430.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">ImageJ software</td>
			<td style="width:169px;"></td>
			<td style="width:187px;"></td>
			<td>Can be downloaded for free from: http://rsb.info.nih.gov/ij/download.html</td>
		</tr>
	</tbody>
</table>

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.

基于使用此协议我们的经验，我们可以放心地推荐为固定细胞ER-线粒体相互作用的可视化和量化这种方法。的在的HuH7肝癌细胞原位接近连接测定-可视ER-线粒体的相互作用，用几对抗体的代表性图像，被示出。如在图2中由荧光显微镜所示，每个红点代表两个目标蛋白质之间的相互作用，以及细胞核显示为蓝色。作为阴性对照，只使用一个一级抗?...

Watch this Scientific Journal Video about Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells at JoVE.com

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

这里，我们描述一个过程中固定的细胞可视化和高灵敏度量化内质网和线粒体之间的内源的相互作用。该协议提供在原位接近连接测定在线粒体相关膜界面靶向1,4,5-三磷酸肌醇受体/葡萄糖调节蛋白75 /电压依赖性阴离子通道/亲环三维复杂的优化。

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), ...

study-endoplasmic-reticulum-mitochondria-interactions-situ-proximity

Research

JoVE Journal

Biology

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

24.5K 次观看.  Lund University. 这里，我们描述一个过程中固定的细胞可视化和高灵敏度量化内质网和线粒体之间的内源的相互作用。该协议提供在原位接近连接测定在线粒体相关膜界面靶向1,4,5-三磷酸肌醇受体/葡萄糖调节蛋白75 /电压依赖性阴离子通道/亲环三维复杂的优化。 

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

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

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

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

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

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, ...

科研

生物学

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质:DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

研究内质网和线粒体相互作用通过

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