作者没有确认。

所有实验程序均由东京都大学体育动物护理与使用委员会（IACUC）批准。根据"实验动物护理和使用 指南"和 "加拿大动物护理委员会实验动物护理和使用指南"，将动物饲养和处理。我们在日本东京都大学前沿健康科学系功能形态学实验室养殖和维护动物。 动物护理和住房<ol><li>从封闭的繁殖群中获得7只小鼠 （3只雌性和4只雄性，重70-90克）。 </li><li>在装有含有纸条的木制巢箱的塑料笼中断奶后分别保存S. murinus （出生后20 d）。将它们保存在常规条件的动物室内:23-27℃，无湿度控制，12:12小时:黑暗循环。供应商业唾液丸和水随意。 </li></ol> 2.组织准备<ol><li>为了制备4％（w / v）多聚甲醛（PFA），将40g PFA溶于1,000mL 0.01M磷酸盐缓冲盐水（PBS，pH7.4）中。在通风柜中，使用涡流进行混合，直到溶液清亮。在4℃下储存4％PFA O / N。 注意:在处理PFA时佩戴适当的个人防护装备（PPE）。 </li><li>灌注并固定动物，用乙醚麻醉，然后腹膜内注射睡眠素（戊巴比妥钠，0.6 mL / kg体重）。 </li><li>动物完全麻醉后，用解剖刀打开腹腔，形成3厘米长的中线腹部切口。在切除下腔静脉用于放血时，将导管逆行插入腹主动脉，在该动脉分叉上方的水平进入髂总动脉。 </li><li>注射嗜睡眠（戊巴比妥）ium，1.0mL / kg体重）。完全安乐死后，用0.01M PBS（pH 7.4）灌注，然后用4％PFA灌装在通风柜中。 </li><li>灌注后，注入2-3mL白色氯丁橡胶胶乳（稀释的白色氯丁橡胶胶乳3:2，用蒸馏水（DW））标记血管。 </li><li>将有关神经和血管的腹部器官，包括肝脏，胆囊，胆总管，十二指肠和胰腺一起提取。 </li><li>将约0.1mL蓝色氯丁橡胶胶乳（向一滴稀释的白色氯丁橡胶胶乳中加入一滴蓝色墨水）注入胆汁以标记胆汁系统。 </li><li>在4℃下用4％PFA固定过夜以进行全套免疫染色。 注意:PFA有毒。避免直接使用PFA。 PFA应用于通风柜。 </li></ol> 3.全身免疫组织化学注意:在整个方案中，包括在洗涤，抗体孵育和着色期间，e样品必须留在钳子上，轻轻地在室温或4℃下摇摆。 <ol><li> 第1天:酶促处理和灭活潜在的内源性过氧化物酶 <ol><li>在适当尺寸的玻璃小瓶中，每个室温下用DW 4x将制备的样品洗涤1小时。将样品轻轻摇匀，以除去PFA。避免在交换溶液时损坏样品。 </li><li>为了制备1％（w / v）的正周期酸，将1g正周期酸溶于100mL的DW中。 </li><li>在室温下将样品在1％正二碘酸中孵育20分钟，以防止任何内在的过氧化物酶反应。 </li><li>通过将0.004g木瓜蛋白酶溶解在100mL 0.025mol / L Tris-HCl缓冲液（pH7.6）中来制备0.004％（w / v）木瓜蛋白酶。 </li><li>在室温下用DW清洗样品10分钟。 </li><li>将样品在新鲜制备的0.004％木瓜蛋白酶中，在37℃下，在温和摇摆的恒温槽中孵育2小时。 </li><li>用D清洗试样W在室温下保持50-60分钟。 </li><li>以4°CO / N将样品储存在4％PFA中。 </li></ol></li><li> 第2天:酶治疗 <ol><li>用DW洗涤存放的样品4次，每次1小时。 </li><li>将样品在新鲜制备的0.004％木瓜蛋白酶中，在37℃下，在温和摇摆的恒温槽中孵育2小时。 </li><li>在室温下用DW洗涤样品50-60分钟。以4°CO / N将样品储存在4％PFA中。 </li></ol></li><li> 第3天:冷冻治疗 <ol><li>按照上述方式，在室温下用DW 4x洗涤储存的样品1小时。 </li><li>通过将2.5g，5g和10g蔗糖分别溶解在100mL的0.01M PBS（0.5g / l）中制备2.5％（w / v），5％（w / v）和10％（w / v） pH 7.4）。 </li><li>将样品浸入2.5％（w / v），5％（w / v）和10％（w / v）蔗糖中30分钟。 </li><li>将样品在-20℃下冷冻30-60分钟，然后在室温下完全解冻;重复周期3x。 </li><li>最佳补充2％Triton X-100（v / v），加入2mL Triton X-100至100mL的0.01M PBS（pH7.4）。 </li><li>将样品以4°CO / N存放在2％Triton X-100中。 </li></ol></li><li> 第4天:初次抗体孵育 <ol><li>通过将0.2g牛血清白蛋白（BSA），0.3mL Triton X-100和0.1g叠氮化钠溶解在100mL 0.01M PBS（pH 7.4）中来制备第一抗体的稀释溶液。 注意:叠氮化钠具有剧毒性。必须避免吸入和接触。穿合适的PPE。 </li><li>在上述稀释缓冲液中稀释第一抗体（NFP）1:600（步骤3.4.1）。在4℃下将标本与NFP抗体孵育3-6天，保持标本轻轻地摇动。较大的标本将需要更长的孵化时间。 </li></ol></li><li> 二次抗体孵育 <ol><li>在PBS中将样品洗涤1次，每次1小时，以除去未结合的一抗。 </l我&gt; <li>通过将0.2g BSA和0.3mL Triton X-100溶解在100mL 0.01M PBS（pH 7.4）中来制备二抗的稀释溶液。 </li><li>在稀释缓冲液中稀释第二抗体（过氧化物酶缀合的，亲和纯化的绵羊抗小鼠IgG-HRP）1:600（步骤3.5.2）。在4℃下将样品与二次抗体孵育3 d，并将样品轻轻晃动在养分器上。 </li></ol></li><li> 着色 <ol><li>通过在通风橱下在100mL 0.05mol / L Tris-HCl缓冲液（pH7.6）中加入0.002g 3,3&#39;-二氨基联苯胺四盐酸盐（DAB）制备DAB着色溶液，将溶液保持在黑暗中。 </li><li>在PBS中4℃洗涤样品1小时。 </li><li>在新制备的DAB着色溶液中加入10μL的30％H 2 O 2 ，并在4°CO / N下孵育3 d，同时在养分器上轻轻晃动。 </li><li>停止pla的反应当达到最佳染色强度时，用0.04％叠氮化钠在PBS中取样。 注意:在此步骤之前，避免使用叠氮化钠，因为它可以抑制辣根过氧化物酶。 </li></ol></li><li> 成像染色的组织 <ol><li>小心地将样品转移到含有PBS的培养皿中（5厘米高，直径10厘米）。使用安装在立体显微镜上的相机拍摄整个安装的图像。 注意:将完全染色的组织成像，完全浸入PBS中的透明玻璃皿上。 </li></ol></li></ol>

<ol>
	<li>Kuratani, S., Tanaka, S., Ishikawa, Y., Zukeran, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+the+hypoglossal+nerve+in+the+chick+embryo+as+observed+by+the+whole-mount+nerve+staining+method.">Early development of the hypoglossal nerve in the chick embryo as observed by the whole-mount nerve staining method.</a> Am. J. Anat. 182 (2), 155-168 (1988).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+the+avian+vagus+nerve+with+special+reference+to+the+morphological+innervation+of+heart+and+lung.">Peripheral development of the avian vagus nerve with special reference to the morphological innervation of heart and lung.</a> Anat. Embryol. (Berl). 182 (5), 435-445 (1990).</li><li>Kuratani, S., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+development+of+avian+trigeminal+nerves.">Peripheral development of avian trigeminal nerves.</a> Am. J. Anat. 187 (1), 65-80 (1990).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=House+musk+shrew,+Suncus+murinus:+a+novel+and+natural+obesity-resistant+animal+model.">House musk shrew, Suncus murinus: a novel and natural obesity-resistant animal model.</a> Obes. Metab. 6, 22-28 (2010).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anatomy+of+innervation+of+the+gallbladder+in+humans+and+Suncus+murinus+with+special+reference+to+morphological+understanding+of+gallstone+formation+after+gastrectomy.">Surgical anatomy of innervation of the gallbladder in humans and Suncus murinus with special reference to morphological understanding of gallstone formation after gastrectomy.</a> World J. Gastroenterol. 13 (14), 2066-2071 (2007).</li><li>Yi, S. 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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anatomy+of+the+innervation+of+the+major+duodenal+papilla+in+humans+and+Suncus+murinus,+from+the+perspective+of+preserving+innervation+in+organ-saving+procedures.">Surgical anatomy of the innervation of the major duodenal papilla in humans and Suncus murinus, from the perspective of preserving innervation in organ-saving procedures.</a> Pancreas. 30 (3), 211-217 (2005).</li><li>Yi, S. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+anatomy+of+the+innervation+of+the+pylorus+in+humans+and+Suncus+murinus,+in+relation+to+the+surgical+technique+for+pylorus-preserving+pancreatoduodenectomy.">Surgical anatomy of the innervation of the pylorus in humans and Suncus murinus, in relation to the surgical technique for pylorus-preserving pancreatoduodenectomy.</a> World J. Gastroenterol. 12 (14), 2209-2216 (2006).</li><li>Yi, S. Q., Ren, K., Kinoshita, M., Takano, N., Itoh, M., Ozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innervation+of+Extrahepatic+Biliary+Tract,+With+Special+Reference+to+the+Direct+Bidirectional+Neural+Connections+of+the+Gall+Bladder,+Sphincter+of+Oddi+and+Duodenum+in+Suncus+murinus,+in+Whole-Mount+Immunohistochemical+Study.">Innervation of Extrahepatic Biliary Tract, With Special Reference to the Direct Bidirectional Neural Connections of the Gall Bladder, Sphincter of Oddi and Duodenum in Suncus murinus, in Whole-Mount Immunohistochemical Study.</a> Anat. Histol. 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作者声明没有利益冲突。

这项工作描述了使用抗神经丝蛋白的抗体对肝外胆道神经支配的可视化的实验程序。该协议根据Kuratani和Tanaka 1，2，3描述的方案进行了改编。 对于这个实验，计划每个实验的时间表，因为整体染色方法持续2-3周。包含组织的容器必须始终在营养器上旋转，除非在冻融过程中。特别是在与一级和二级抗体一起孵育的同时，将样品置于冷藏室中，同时在养分器?...

房子麝香rew，，，属于食虫纲和家Sor科。这种小型哺乳动物分布在整个东南亚，栖息在靠近人类栖息地或牛栏的房屋和草地上。与其他实验室动物（如小鼠，大鼠和兔5）相比，该物种具有与人类相似的一般形态特征。以前，使用具有S. murinus神经丝蛋白（NFP）的外周神经元标记的全蛋白免疫染色来标记胰腺6 ，主要十二指肠乳头7 ，幽门8 ，胆囊5和肝外胆道9中的外周神经。 NFP是成熟神经元细胞骨架的一部分的大分子复合物。神经丝相关蛋白介导NFP和Zygosome之间的相互作用。酶功能和接头蛋白的结构均受NFP 10调控。 NFP复合物由三种多肽组成:NF-L（70kDa），NF-M（150-160kDa）和NF-H（200kDa）。 NFP可以在中枢神经系统和外周神经系统的神经元轴突中发现。已经显示抗人NFP抗体标记中枢和周围神经系统的轴突。解剖和电生理调查表明，括约肌，胆囊和近端胃肠道连接11,12,13,14。然而，他们的神经元连接的形态特征尚未被定义。 这项工作表明使用全套免疫组织化学染色方法来标记耻骨球菌的神经支配使用NFP抗体。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">orthoperiodic acid</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">162-00732</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">papain</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">164-00172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin (BSA)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">010-15131</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium azide</td>
			<td style="width:71px;">Nacalai Tesque</td>
			<td style="width:119px;">312-33</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neurofilament protein (NFP) antibody</td>
			<td style="width:71px;">Dako</td>
			<td style="width:119px;">M0762, lot 089, clone: 2F11</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">peroxidase-conjugated affinity-purified sheep anti-mouse IgG-HRP</td>
			<td style="width:71px;">MBL</td>
			<td style="width:119px;">Code 330</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrochloride (DAB)</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">349-00903</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">imidazole</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I-0125</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a whole-mount immunohistochemical method to study the innervation of the biliary tract in suncus murinus

本文详细描述了全基因免疫组织化学染色方法，使用神经丝蛋白抗体标记阳性丝猴（ S. murinus）胆道神经支配 &#160; ）。首先从S. murinus中切下标本并固定在4％多聚甲醛（PFA）中。其次，进行酶处理和潜在的内源性过氧化物酶失活。然后将标本暴露于一抗，抗神经丝蛋白抗体3-6天。然后与与辣根过氧化物酶缀合的二抗孵育。通过使样品与3,3&#39;-二氨基联苯胺（DAB）底物反应来显示显色反应。该方法可用于分析所有内脏器官的神经支配。此外，该方案还可以适应于测试其他神经元抗体，但优化抗体应先做好。这种方法最初由Kuratani和Tanaka 1，2，3引入。

图 1-4显示了S. murinus肝外胆道中NFP阳性神经纤维的典型结果。抗NFP的抗体可重复标记肝外胆管（ 图1 ），胆囊（ 图2 ），上胆管（ 图3 ）和十二指肠乳头区域（ 图4 ）的整个图像中的神经支配，具有高特异性和最小背景。 <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus at JoVE.com

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

一种全身免疫组织化学方法，可以显示Suncus murinus肝外胆道中的神经丝蛋白表达。在这里介绍。该方案可用于分析小鼠或其他物种中所有内脏器官的神经支配。

使用全身免疫组织化学方法研究胆道的神经支配<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

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Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

6.7K 次观看.  Tokyo Metropolitan University. 一种全身免疫组织化学方法，可以显示Suncus murinus肝外胆道中的神经丝蛋白表达。在这里介绍。该方案可用于分析小鼠或其他物种中所有内脏器官的神经支配。 

视频: Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

使用全山原位杂交分子和有机体生物学链接

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析:一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

使用全身免疫组织化学方法研究胆道的神经支配

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此视频中的章节

测试的速度和运动方向的视觉灵敏度在蜥蜴

双整装原位杂交技术在早期鸡胚

整装在鸡的抗体染色方法

斑马鱼整装高分辨率双荧光原位杂交

嗨 - C：一个方法研究基因组的三维架构。

使用全山原位杂交分子和有机体生物学链接

全安装胚胎肢体皮肤血管的免疫组织化学分析:一个模型系统，研究在胚胎血管分支形态发生

全山原位杂交E8.5，E11.5小鼠胚胎

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism