我们要认识到，在发展本议定书Dörthe利希，珍妮回合和安德鲁马拉的贡献。 NICHD的，美国癌症协会和March of Dimes的提供的研究支持。

1。固定<ol><li>修复的胚胎在4℃过夜℃，4％多聚甲醛（PFA）的PBS。 </li><li>删除修复，并在室温（RT）洗2 × PBS，每次5分钟。 </li><li>在PBS手动dechorionate胚胎在玻璃抑郁使用钟表匠钳板。 dechorionation后，胚胎转移使用火抛光的玻璃巴斯德吸管，因为他们可能会粘到聚丙烯移液器。 </li><li>转移胚胎通过一系列的25％，50％和75％甲醇的PBS，每次5分钟。 </li><li>用100％的甲醇液体，孵化5分钟，然后与新鲜甲醇代替。 </li><li>在-20 ° C放置至少一小时的胚胎。 （我们通常孵化的胚胎过夜。原位杂交技术标准工作了一年多的储存的胚胎，但我们还没有研究是否高分辨率的荧光原位杂交长时间存放丢失。） </li><li>每次5分钟，75％，50％，25％甲醇在室温下PBST清洗胚胎。 5分钟在室温下在PBST洗两次。 </li><li>在4％PFA在室温下用PBS再次修正为20分钟。 </li><li> 5分钟在室温下在PBST洗两次。 </li></ol>注：关于煤灰： 我们存放在-20℃的4％PFA（见试剂表）分装。初步固定，我们只用煤灰以前从未有过解冻。随后的录制品中，我们经常用煤灰先前已解冻。初步固定，似乎是与此协议的成功染色关键。 2。蛋白酶和POSTFIXATION <ol><li>精华与蛋白酶K（5μg/ml在PBST）在3至12分钟，以通透的胚胎RT。 （培养时间取决于胚胎，作为年轻的阶段是比较敏感的年龄，也取决于一批酶）。somitogenesis阶段的胚胎，我们通常通透3-4分钟。在此孵化，我们打下的离心管中，在其一侧。 </li><li>简述在PBST冲洗，并为5分钟，在PBST洗一次。 </li><li>在4％PFA在室温下用PBS再次修正为20分钟。 </li><li>洗两次，每次5分钟，在室温下PBST。 </li></ol> 3。预杂交<ol><li>在65 º C孵育5分钟的胚胎HYB -。 </li><li> Prehybridize在HYB至少1小时+ 65 ° C。 </li></ol> 4。杂交<ol><li>取出所有的preHYB 50μL，但一定要保持完全淹没的胚胎。 </li><li>每个riboprobe 1-2μL（digoxygenin和荧光标记的riboprobes）和混合胚胎，轻轻弹管。探头的数量通常是1 -2μL从20μL探针的合成反应。 </li><li>管，荧光光敏感，应在铝箔包裹或以其他方式接触到最小光从这一点向前。 </li><li>在65 ° C孵育过夜的胚胎</li></ol> 5。探头撤除请注意，从这一点向前缺乏清洁剂的解决方案。消除洗涤剂似乎可以帮助染色反应，但不会导致胚胎变得相当粘。 <ol><li>取出的riboprobe。 </li><li>在65 º C 50％formamide/2xSSC洗2 × 30分钟。 </li><li>在65 º C 2 × SSC洗15分钟。 </li><li>洗30分钟，65 º C在0.2 × SSC。 </li></ol> 6。抗荧光抗体孵育<ol><li>在室温下至少1小时座1X马来酸缓冲液加2％阻断试剂（见试剂表）的解决方案500μL。 </li><li>添加在封闭液1:500稀释的抗荧光素过氧化物酶抗体，由罗氏公司提供，。 </li><li> 4℃孵育过夜在此孵化，我们打下的离心管中，在其一侧。 </li><li> 1X马来酸缓冲液洗4 × 20分钟。 5分钟的PBS洗两次。 </li></ol> 7。检测荧光标记的探针<ol><li> TSA加荧光液孵育30-60分钟。 （染色液前离心TSA的基板反应，淡化酪胺试剂Perkin Elmer公司扩增稀释剂缓冲区1:50）在此孵化，我们打下的离心管中，在其一侧。反应时间必须为每个探头凭经验确定。不幸的是，染色反应，不能直观地监测为基材的荧光，会看到整个染色反应无处不在的绿色荧光。 </li><li> 30％，50％，75％和100％甲醇的PBS洗，每次10分钟。 </li><li>在解决方案，在甲醇30分钟的1％ 的 H 2 0 2孵育灭活过氧化物酶。 </li><li> 75％，50％和30％甲醇的PBS洗涤，每次10分钟。在PBS各10分钟，然后洗两次。重要的是，所有的大都会hanol被删除。 </li></ol>酪胺信号放大的注意事项： 我们一直在使用Perkin Elmer公司TSA试剂盒。我们发现，Alexa的酪胺基板染色以及使用Perkin Elmer公司扩增稀释剂缓冲区，但我们还没有成功使用的染色与Invitrogen公司/分子探针试剂盒提供的缓冲区。最后，我们发现，随后甲醇/ H 2 O 2处理淘汰，而荧光素和Alexa - 647不受影响Cy5的荧光。 Cy3标记也可能会受到不利影响的甲醇/ H 2 O 2处理，因为它是结构相关的，以Cy5的。出于这个原因，Cy3和Cy5 TSA的反应应该只被用于第二双荧光染色原位协议反应。 8。抗- Digoxygenin抗体孵育<ol><li>座在室温下至少1小时的胚胎，再次在1X马来酸缓冲液加2％阻断试剂的解决方案。 </li><li>添加提供由罗氏公司在上述封闭液在1:1000稀释的抗地高辛的POD抗体</li><li> 4℃孵育过夜在此孵化，我们打下的离心管中，在其一侧。 </li><li> 1X马来酸缓冲液洗4 × 20分钟。 5分钟的PBS洗两次。 </li></ol> 9。 Digoxygenin标记探针检测<ol><li>孵育（上下TSA的衬底旋转，然后才作出染色溶液反应，淡化酪胺试剂放大稀释剂缓冲区1:50）30-60分钟，在TSA的加Cy5的解决方案在此孵化，我们打下的离心管中，在其一侧。反应时间必须为每个探头凭经验确定。 </li><li> 10分钟，在PBST清洗三次。 </li></ol> 10。碘化丙啶染色<ol><li> 5分钟，在2 × SSC洗两次。 </li><li>孵育30分钟，胚胎在37 ° 50μL2 × 10μLRNA酶（终浓度为100μg/ml的）SSCT与C。 </li><li>在2 × SSC在室温下每3分钟清洗6次。 </li><li>为8分钟，在2 × SSC溶液330μg/ml碘化丙啶染色的胚胎。 </li><li>在2 × SSC在室温下每3分钟清洗6次。 </li><li>修复了20分钟在4％PFA在室温下用PBS。 </li><li>洗两次，每次5分钟，在室温下PBST。 </li></ol> 11。 MOUNTING <ol><li>孵育10分钟，每个在25％和50％甘油在PBST的胚胎。清除75％的甘油，4℃过夜</li><li>我们解剖和deyolk胚胎和平板安装在显微镜幻灯片。蛋黄可产生显着的背景荧光。剥离是容易得多的胚胎后，已清除gylcerol ovenight。 </li></ol>解决方案： <table border="1"><tbody><tr><td> PBST </td><td> PBS加0.1％吐温</td><td></td></tr><tr><td> SSCT </td><td> SSC加0.1％吐温</td><td></td></tr><tr><td> HYB - </td><td> 50％甲酰胺 5xSSC 0.1％Tween - 20的</td><td></td></tr><tr><td> HYB + </td><td> HYB - 5mg/ml torula（酵母）RNA 50μg/ml肝素</td><td style="width: 300px;"> torula蛋白酶K消化的RNA与RNA准备随后苯酚，苯酚 - 氯仿，氯仿extraction.The RNA沉淀溶解于DEPC处理过的水。 </td></tr><tr><td> 1个马来酸缓冲</td><td>马来酸150MM，100MM氯化钠（pH值7.5） </td><td></td></tr></tbody></table><img src="/files/ftp_upload/1229/Brend_Holley_Fig1.png" alt="图1" width="600" height="688" /> 图1。整装代表结果双荧光原位杂交。 （AC）。图像显示，单焦部分通过对斑马鱼的胚胎在10体节期后区。据认为是与前顶端背。原子核与碘化丙啶染色为蓝色。 （一）deltaC mRNA的检测，使用digoxygenin标记riboprobe和TSA - Cy5的试剂。 （二）mRNA的转录从HER1基因检测用荧光标记的riboprobe和TSA荧光素试剂。 （三）合并，绿色和红色通道，明确标识不同的或重叠的两个基因的基因表达的区域。 （D，E）HER1表达的细节，展示了此过程的高分辨率。 mRNA的亚细胞定位，可以清楚地看出端倪。箭头指示表现活跃转录的一个细胞，发现在细胞核染色点。箭头指示一个细胞的mRNA的细胞质定位。 （F，G）HER1和deltaC的双重染色显示细胞转录基因（白箭头），或任一基因分开（红色和绿色箭头） 。

<ol>
	<li>Schulte-Merker, S., Ho, R. K., Herrmann, B. G., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+product+of+the+zebrafish+homologue+of+the+mouse+T-gene+is+expressed+in+nuclie+of+the+germ+ring+and+the+notochord+of+the+early+embryo.">The protein product of the zebrafish homologue of the mouse T-gene is expressed in nuclie of the germ ring and the notochord of the early embryo.</a> Development. 116, 1021-1027 (1992).</li><li>Jowett, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+in+situ+hybridization+techniques+in+zebrafish.">Double in situ hybridization techniques in zebrafish.</a> Methods. 23, 345-358 (2001).</li><li>J&#252;lich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beamter/deltaC+and+the+role+of+Notch+ligands+in+the+zebrafish+somite+segmentation,+hindbrain+neurogenesis+and+hypochord+differentiation.">beamter/deltaC and the role of Notch ligands in the zebrafish somite segmentation, hindbrain neurogenesis and hypochord differentiation.</a> Dev Biol. 286, 391-404 (2005).</li><li>Mara, A., Schroeder, J., Chalouni, C., Holley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming,+Initiation+and+Synchronization+of+the+Segmentation+Clock+by+deltaD+and+deltaC.">Priming, Initiation and Synchronization of the Segmentation Clock by deltaD and deltaC.</a> Nat Cell Biol. 9, 523-530 (2007).</li><li>Lecuyer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+mRNA+localization+reveals+a+prominent+role+in+organizing+cellular+architecture+and+function.">Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function.</a> Cell. 131, 174-187 (2007).</li></ol>

这里提出的协议的工作原理以及与染色30-45分钟后，在一个典型的碱性磷酸酶介导的反应，让一个干净的强烈信号的探针。执行的荧光原位杂交协议之前，我们总是考验我们的探头，使用标准的非荧光协议（探针合成协议提供补充材料）。使用较弱的探针时，我们曾成功与荧光原位杂交协议，但它可能在某些情况下可能。我们已经成功地结合起来，与β- catenin的3免疫定位的荧光原位杂交协...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Paraformaldehyde 16% solution, EM Grade</td>
<td>&nbsp;</td>
<td>Electron Microscopy Services</td>
<td>15710</td>
<td>Dilute to 4% in 1.33x PBS (final 1x). Store in 2ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Proteinase K, recombinant PCR grade</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115879001</td>
<td>Make a 20mg/ml stock solution in water. Store in 1ml aliquots at &#x2013;20&deg;C</td>
</tr>
<tr>
<td>Blocking Reagent</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11096176001</td>
<td>Make a 10% stock solution in 1x maleic acid buffer. Store in 50ml aliquots at &#x2013;20&deg;C. Stable over multiple freeze/thaws.</td>
</tr>
<tr>
<td>Anti-Fluorescein-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11426346910</td>
<td>Aliquot and store at -20&deg;C. Keep one working aliquot at 4&deg;C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-POD, Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11207739910</td>
<td>As above.</td>
</tr>
<tr>
<td>TSA Plus Fluorescein system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL741001KT</td>
<td>Prepare each vial of TSA reagent only when needed. Dissolve in 60&mu;l DMSO. Store at 4&deg;C.</td>
</tr>
<tr>
<td>TSA Plus Cyanine5 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL745001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Plus Cyanine3 system</td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>NEL744001KT</td>
<td>As above</td>
</tr>
<tr>
<td>TSA Kit #16 AlexaFluor647 tyramide (plus HRP-goat-anti-rabbit IgG)</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>T20926</td>
<td>Dissolve tyramide reagent in 150&mu;l DMSO, aliquot and store at &#x2013;20&deg;C.</td>
</tr>
<tr>
<td>RNase, DNase free</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11119915001</td>
<td>Supplied as 500&mu;g/ml solution</td>
</tr>
<tr>
<td>Propidium Iodide</td>
<td>&nbsp;</td>
<td>Molecular Probes /Invitrogen</td>
<td>P-3566</td>
<td>Supplied as 1mg/ml solution in water.</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish whole mount high-resolution double fluorescent in situ hybridization

整装<em&gt;原位</em&gt;杂交是发育生物学中最广泛使用的的技术之一。在这里，我们提出了一个高分辨率的双荧光<em&gt;原位</em&gt;为一个单一的基因精确的表达模式分析和确定两个基因的表达域重叠的杂交的协议。该协议是一个标准的修改后的版本<em&gt;原位</em&gt;杂交利用碱性磷酸酶和NBT / BCIP显和快速红如基板<sup&gt; 1,2</sup&gt;。该协议利用酪胺信号放大（TSA）的标准di​​goxygenin和荧光标记的探针<sup&gt; 3</sup&gt;。市售的TSA套件允许各种核污渍，如碘化丙啶，或荧光蛋白免疫组织化学，灵活的荧光发射的实验设计，从绿色到远红光可结合使用。 TSA产生的反应的荧光底物，快速共价键结合的基团，通常的酪氨酸残基在紧邻的标记的反义riboprobe，。染色模式，亚细胞定位的mRNA高分辨率使用激光扫描共聚焦显微镜可观察到<sup&gt; 3,4</sup&gt;。在染色体位点，人们可以观察到新生的成绩单，分清细胞核和细胞质染色和可视化等皮质定位的mRNA模式。在研究<em&gt;果蝇</em&gt;表明，大约有70％的mRNA表现经常与编码的蛋白质的功能相关的亚细胞定位的具体模式<sup&gt; 5</sup&gt;。结合计算机辅助三维共焦数据集重建时，我们的协议允许的mRNA的亚细胞分布在整个脊椎动物胚胎的决议的详细分析。

Watch this Scientific Journal Video about Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization at JoVE.com

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

整装原位杂交是发育生物学中最广泛使用的技术之一。在这里，我们提出一个高分辨率的双荧光原位杂交协议分析一个单一的基因精确的表达模式，并确定两个基因的表达域重叠。我们包括核反击一个碘化丙啶染色，以突出组织机构。

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 ...

zebrafish-whole-mount-high-resolution-double-fluorescent-situ

Research

JoVE Journal

Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

23.1K 次观看.  Yale University. 整装原位杂交是发育生物学中最广泛使用的技术之一。在这里，我们提出一个高分辨率的双荧光原位杂交协议分析一个单一的基因精确的表达模式，并确定两个基因的表达域重叠。我们包括核反击一个碘化丙啶染色，以突出组织机构。

视频: Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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 ...

科研

生物学

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

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 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 ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

单细胞基因转录的RNA荧光分析<em在原位</em&gt;杂交（FISH）

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...