Name: whole mount in situ hybridization of e8.5 to e11.5 mouse embryos
Uploaded: 2011-10-10T12:00:00
Description: Watch this Scientific Journal Video about Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos at JoVE.com

这项工作是由国立卫生研究院拨款R21MH082360（BGC）和R01HD056315（NRM）以及佐治亚大学的支持。

1。通过体外转录的Riboprobe代<ol><li> 准备在体外转录模板的PCR产物 。 <ol><li>噬菌体在其5&#39;端转录启动子序列设计PCR引物。 注：启动子序列的意义链的PCR引物5&#39;端，将抄录感探头，并添加到启动子序列的反义PCR引物5&#39;端，将合成的反义探针1-3。我们并没有发现任何差异之间的模板，其中包含只有核心启动子序列与模板包含核心启动子加上额外的5&#39;序...

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在本议定书中所描述的方法已经适应从不同来源和整装E8.5 - E11.5日龄小鼠胚胎进行了优化。整个装载在脊椎动物胚胎原位杂交方法最早出现在20世纪90年代初2,5-12。该协议被改编，主要是从非洲爪蟾胚胎 7,8以及鼠标 2,11开发的方法。在我们的协议中，很大的重点放在探头严格的质量评估。仔细注意产生高品质和高比活性的RNA探针在这个协议中最早的步骤确定将...

<table border="1"><tbody><tr><th>名称</th><th>类型</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>地高辛- 11 -尿苷-5&#39; - 三磷酸</td><td>试剂</td><td>罗氏公司</td><td> 11209256910 </td><td></td></tr><tr><td>核苷三磷酸集</td><td>试剂</td><td>罗氏公司</td><td> 11277057001 </td><td></td></tr><tr><td>放射性标记的RNA纯化的快速离心柱</td><td>供应</td><td>罗氏公司</td><td> 11274015001 </td><td></td></tr><tr><td> T7 RNA聚合酶</td><td>试剂</td><td>罗氏公司</td><td> 10881767001 </td><td></td></tr><tr><td> SP6 RNA聚合酶</td><td>试剂</td><td>罗氏公司</td><td> 10810274001 </td><td></td></tr><tr><td> T3 RNA聚合酶</td><td>试剂</td><td>罗氏公司</td><td> 11031163001 </td><td></td></tr><tr><td> CTP中，[α- 32P] - 800Ci/mmol 10mCi/ml，250μCi </td><td>试剂</td><td> Perkin Elmer公司</td><td> BLU008X250UC </td><td></td></tr><tr><td> DNA酶我重组，无RNase </td><td>试剂</td><td>罗氏公司</td><td> 4716728001 </td><td></td></tr><tr><td>尿素，无色至白色结晶或结晶性粉末</td><td>试剂</td><td>费舍尔</td><td> BP169 - 500 </td><td></td></tr><tr><td>凝胶上样缓冲液</td><td>试剂</td><td> Ambion公司</td><td> AM8547 </td><td></td></tr><tr><td> Hybond N +，Amersham公司</td><td>供应</td><td> GE医疗集团</td><td> RPN82B </td><td></td></tr><tr><td>紫外线Stratalinker 2400 </td><td>设备</td><td> Stratagene公司</td><td></td><td></td></tr><tr><td>焦碳酸二乙酯</td><td>试剂</td><td>西格玛</td><td> D5758 - 100ML </td><td></td></tr><tr><td>肝素钠</td><td>试剂</td><td>阿库罗斯福冈</td><td> 41121-0010 </td><td></td></tr><tr><td> 30％过氧化氢在水中</td><td>试剂</td><td> Fisher Scientific则</td><td> BP2633 - 500 </td><td></td></tr><tr><td> Gluteraldehyde，8％的EM级</td><td>试剂</td><td> Polysciences </td><td> 0 / 710 </td><td>根据制造商的指示慎用</td></tr><tr><td>阻断试剂</td><td>试剂</td><td>罗氏公司</td><td> 11096176001 </td><td>在-20 ° C到5％的股票和存储</td></tr><tr><td>蛋白酶K </td><td>试剂</td><td>罗氏公司</td><td> 3115852001 </td><td></td></tr><tr><td>核糖核酸酶A </td><td>试剂</td><td>罗氏公司</td><td> 10109142001 </td><td>请为10毫克/毫升的股票，并存放于-20 ° C </td></tr><tr><td>核糖核酸酶T1 </td><td>试剂</td><td>罗氏公司</td><td> 10109193001 </td><td></td></tr><tr><td>超纯甲酰胺</td><td>试剂</td><td> Invitrogen公司</td><td> 15515-026 </td><td></td></tr><tr><td>盐酸左旋咪唑</td><td>试剂</td><td>仁川Biomedicals。公司</td><td> 155228 </td><td></td></tr><tr><td> Ⅵ型圆酵母，核糖核酸</td><td>试剂</td><td>西格玛</td><td> R6625 </td><td>酚/氯仿多次提取和沉淀，重悬于DEPC - dH2O，并储存在-20 ° C </td></tr><tr><td>抗地高辛- AP Fab片段</td><td>试剂</td><td>罗氏公司</td><td> 11093274910 </td><td></td></tr><tr><td> BM紫AP的底物，沉淀。准备使用的解决方案。 </td><td>试剂</td><td>罗氏公司</td><td> 11 44​​2 074 001 </td><td></td></tr><tr><td> 4ML，清除，封闭顶部，存储瓶便利工具包</td><td>供应</td><td>国家科学技术进步奖</td><td> B7800 - 2 </td><td></td></tr><tr><td>摇ñ烘烤杂交炉</td><td>设备</td><td>布克尔科学</td><td> 136400 </td><td></td></tr><tr><td>当然，TEK活检</td><td>供应</td><td> Fisherbrand </td><td> 15 - 200 - 402C </td><td></td></tr><tr><td> Paraplast加组织包埋剂</td><td>试剂</td><td> Fisherbrand </td><td> 23-021-400 </td><td></td></tr><tr><td>剥离一个单程的一次性塑料组织包埋模具</td><td>供应</td><td> Polysciences </td><td> 18646A </td><td></td></tr><tr><td> Colorfrost加显微镜幻灯片</td><td>供应</td><td> Fisherbrand </td><td> 12-550-17 </td><td></td></tr><tr><td>核固红</td><td>试剂</td><td>西格玛</td><td> N8002 </td><td></td></tr><tr><td> Cytoseal 60 </td><td>试剂</td><td>理查德艾伦科学</td><td> 8310-16 </td><td></td></tr></tbody></table>

whole mount in situ hybridization of e8.5 to e11.5 mouse embryos

整装原位杂交，是一个非常翔实的办法确定胚胎中的基因表达模式。 原位杂交程序冗长，技术上与多个重要步骤，集体作出贡献的最终结果的质量要求，该协议详细描述了优化探针标记和性能的几个关键质量控制步骤。总的来说，我们的协议提供了详细的说明必要的关键步骤，可重复获得高质量的结果。首先，我们描述了一代digoxygenin（DIG）标记的RNA探针通过在体外经PCR产生的DNA模板转录。我们描述了三个关键的质量控制检测，以确定的数量，完整性和辛标记探针的具体活动。这些步骤是重要的生成足够的灵敏度探测器检测内源性的mRNA在整个小鼠胚胎。此外，我们描述的固定和储存E8.5 - E11.5日龄小鼠胚胎原位杂交的方法。然后，我们描述了详细的方法有限蛋白酶K消化杂交条件的细节，杂交后洗涤和核糖核酸酶处理，除去非特异性探针杂交的水化胚胎。 AP -共轭抗体用于可视化探针，并揭示了内源性转录的表达模式。代表实验成功和典型的不理想的实验结果显示。

Watch this Scientific Journal Video about Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos at JoVE.com

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

这整个坐骑<em&gt;原位</em&gt;杂交协议的讨论，以确保在E8.5 - E11.5日龄小鼠胚胎的基因表达研究的重复性高品质的成果的关键步骤。

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 ...

Research

JoVE Journal

Biology

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

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

整装在鸡的抗体染色方法

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

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 ...

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

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

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 ...

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

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

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

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

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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

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

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 ...