这项工作是支持的玛格丽特M. Alkek基金会RHF。

第1部分：固定的胚胎<ol><li>填充与冰冷的DEPC - PBS清扫菜。置于冰上。打开用镊子轻敲壳蛋和删除壳件。 </li><li>用镊子取出厚厚的白蛋白。卵黄囊倾斜粗钳，使胚胎朝上。 </li><li>用剪刀，切割围绕胚胎的卵黄囊平方米。 </li><li>使用勺子，取出胚胎从卵黄和冰冷的DEPC - PBS。 </li><li>在显微镜下，删除膜和蛋黄，并转移到固定菜。 </li><li>牵制，除去DEPC - PBS。 DEPC - PBS在4％煤灰更换和修复O / N在4 ° C。 </li><li>删除固定液。更换与冰冷的DEPC - PBS。使用钝年底microcapillary针或显微切割刀，穿孔的神经系统和心腔，防止探头诱捕。 </li><li> 2X 10分钟DEPC - PBS洗0.1％Tween - 20的（PBT）的。脱水10分钟每25％，50％，75％甲醇在PBT。两次在100％甲醇脱水胚胎。在甲醇储存在-20 ° C。 </li></ol>第2部分：预杂交<ol><li>补充水分胚胎75％，50％和25％甲醇在PBT中的每个10分钟。在PBT洗净2X 10分钟。 </li><li>同时，准备冰冷的固定液（4％多聚甲醛在PBT）。替换蛋白酶K溶液（3毫升/瓶; [5克/毫升]最后在PBT）PBT。确保整个小瓶暴露蛋白酶K溶液轻轻滚动小瓶;故障排除的曝光时间。 </li><li>使用核糖核酸免费巴斯德吸管删除蛋白酶K，添加固色剂（2毫升）。整整20分钟，立即放在冰上。 </li><li>所有RT洗上nutator执行。洗2X 10分钟，在PBT和1x在PBT 10MN含有50％的杂交缓冲液。 </li><li>在杂交缓冲液清洗1X。孵育65 °彗星在4-5小时的杂交缓冲液2毫升。 </li></ol>第3部分：与探针的杂交胚胎<ol><li>挖掘和FITC耦合探针合成是在附录中描述。预计将提供一个强有力的信号探头，应synhesized FITC含核苷酸;较弱的探头应与辛含核苷酸合成。 </li><li> Prewarm探针在杂交缓冲液（500毫微克/毫升），使用2 ml小瓶。及时更换探头解决方案的杂交缓冲液。不要让胚胎干。孵育16小时，在65 ° C。 </li><li>替换探针杂交缓冲液，2洗，30分钟在65 ° C。在杂交缓冲液/ MABT中清洗15分钟（1 / 1 V / V /）在65 ° C。 </li></ol>第4部分：辛抗体孵育和显色反应<ol><li>洗净MABT 2X20万。在2％BBR / MABT清洗1小时。洗5小时，在2％BBR/20％HISS / MABT（封闭液）。孵育封闭液稀释1:2000辛抗体，O / N在4 °上nutator彗星。 </li><li>洗3X 10分钟在MABT，3X MABT每1小时。 </li><li>洗净NTMT 2X 20分钟。加入200ul NBT / BCIP底物10毫升NTMT。立即从小瓶NTMT和更换1-2毫升NBT / BCIP显缓冲区。在黑暗的地方。 20分钟后，显示器的色彩反应，并在此后每隔20分钟。 </li><li>显色反应（应该出现蓝色），在PBS洗10分钟，针用PBS填充的固定菜，并取代与4％的PBS PFA解决方案。修正/ O在4 ° C。 </li><li>转移到新的闪烁瓶中。 PBST洗涤（PBS 1％吐温20）2X10万。在MABT 2X 10分钟洗净。 </li><li>在63 ° C，在MABT 30分钟的孵育</li></ol>第5部分：FITC标记抗体孵育和显色反应<ol><li>洗2倍的MABT 10分钟，1小时2％，5小时的封闭液BBR / MABT </li><li>孵育在碱性磷酸酶偶联抗FITC抗体（1:500）的O / 4个N ° C。 </li><li>洗3X 10分钟在MABT，3X MABT每1小时。 </li><li>在TBS的pH值8.45与0.1％Tween - 20的（TBST）2X 20分钟洗净。 </li><li>准备在TBST使用试剂盒提供的试剂1,2和3的矢量红溶液（5毫升）。立即从小瓶TBST和替换矢量红缓冲区。在黑暗的地方。监控显色反应后40分钟，30分钟，其后每隔。 </li><li>下列颜色反应（应该会出现红色），将胚胎转移，以PBS。 </li></ol>第6部分：固定和加工<ol><li>转移到含PBS的固定菜，和4％PFA固定在室温或O / N为20分钟，在4 ° C。 </li><li>在PBS，2个10分钟洗净。摄影过程中，转移至20％甘油的PBS（这将使半透明的胚胎）。要创建一个会议厅，切2条PVC胶带，幻灯片上叠加。切方在使用刀片中心。去掉方使用产钳。 </li><li>蜡切片的过程中，转让，以PBS冲洗2X 10分钟，脱水甲醇梯度（25％，50％，75％和100％的PBS，每10分钟）。 </li><li>丙醇，3MN更换。 1,2,3,4 - 四氢更换，20分钟之后Histoclear（2个1小时）。更换histoclear /蜡1 / 1（V / V）60 ° C时为1小时。替换一个过程蜡为组织学。 </li></ol>第7部分：解决方案杂交缓冲液中，保存在-20 ° C的猎鹰，50毫升25毫升甲酰胺; 3.25毫升（20xSSC）; 0.5毫升（0.5M EDTA）; 1毫升（10％Tween - 20的）; 2.5毫升（1％SDS） 125 UL（20mg/ml tRNA的）; MABT 100ul（50毫克/毫升的肝素），准备100MM马来酸，pH值至7.5氢氧化钠颗粒。 150MM氯化钠和0.1％Tween - 20的。 NTMT（在使用前），50毫升：1毫升（5M氯化钠）; 2.5毫升（2M的Tris - HCl pH9.5）; 1.25毫升（2M氯化镁2）; 5毫升（10％Tween - 20的）。 TBST：0.1M的Tris - HCl，pH值8.45，0.15米，氯化钠0.1％Tween - 20的。 第8部分：故障排除<table border="1"><tbody><tr><th>问题</th><th>原因</th><th>补救</th></tr><tr><td valign="top">胚胎蜷缩</td><td valign="top">脱水/补液不足</td><td valign="top">维持在10分钟的间隔连续脱水/补液步骤</td></tr><tr><td valign="top">有没有探头的信号</td><td valign="top">探头是退化RNase的污染小瓶</td><td valign="top"> 1％琼脂糖凝胶上运行的探头确保整个小瓶蛋白酶K溶液暴露在第2步</td></tr><tr><td valign="top">探头是标签的心腔神经管</td><td valign="top">探头被困住</td><td valign="top">在第2步，确保所有空腔穿孔采用显微切割刀或microcapillary </td></tr><tr><td valign="top">胚胎解体以下杂交（步骤3） </td><td valign="top">蛋白酶K一步离开太久杂交温度过高</td><td valign="top">蛋白酶K不应该离开不再比1mn（阶段3和4），2分钟（第一阶段5至7），3分钟（8-10期），4分钟（第一阶段11-13）不要在 T O杂交更高超过摄氏65度</td></tr></tbody></table>第9部分：代表结果代表胚胎如下：（一）胚胎（阶段HH7），是标示与DIG - SOX探头（蓝色）和一个FITC -δ- 1的探头（红色）。 （二）胚胎（阶段HH8）都标有一挖，PAX6探头（蓝色）和FITC - SHH探头（红色）。 NF，神经倍，ANP，前神经板，PSM，presomitic中胚层，N，NT，脊索，神经管）。从博士实验室的探头礼物。 C.塔宾，古尔丁，D.恩里克，和J.布里斯科。 <img src="/files/ftp_upload/old/904_2008_10_20_1/904_Figure-01.png" alt="" width="303" height="300" />

<ol>
	<li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. 406, 74-78 (2000).</li><li>Rodr&#237;guez Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A., Izpis&#250;a Belmonte, J. 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+novel+Cer-like+protein+Caronte+mediates+the+establishment+of+embryonic+left-right+asymmetry.">The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry.</a> Nature. 401, 243-251 (1999).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Kawakami, M., Nakanishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+an+endogenous+PKA+inhibitor,+PKIalpha,+in+organizing+left-right+axis+formation.">The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation.</a> Development. 128, 2509-2515 (2001).</li><li>Basch, M. L., Bronner-Fraser, M., Garcia-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+the+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of the neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li></ol>

The authors have nothing to disclose.

2色整装原位杂交方法是用来确定空间和时间的基因表达模式，使用一个或两个基因的兴趣。应用领域包括新基因的基因表达模式的评估 （如1,2，以及以下侮辱基因表达的变化（3 胚胎操作 ，珠4，RNA 或DNA电构造5）。

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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>Eggs</td>
<td>Animal</td>
<td>Charles River </td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Hybaid</td>
<td>Micro-4</td>
<td>&nbsp;</td>
<tr>
<td>Adams Nutator orbital mixer</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-062</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Spoon</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10370-18</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Keep pins together using a magnetic stirrer;</td>
</tr>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Tween-20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1379-100ML</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P2308-5MG</td>
<td>Stock at 10 mg/ml in depc-H2O in 10ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>Formamide, deionized</td>
<td>Reagent</td>
<td>Ambion</td>
<td>9342</td>
<td>&nbsp;</td>
<tr>
<td>Yeast tRNA-25 mg</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15401-011</td>
<td>Stock at 20 mg/ml in depc-H2O in 125ul aliquots. Store at -20C</td>
</tr>
<tr>
<td>Heparin Sodium salt</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4784-250MG</td>
<td>Stock at 50 mg/ml in depc-H2O in 100ul aliquots. Store at -20C.</td>
</tr>
<tr>
<td>SSC x20 pH5.5</td>
<td>Reagent</td>
<td>Fisher</td>
<td>PR-V4261</td>
<td>&nbsp;</td>
<tr>
<td>EDTA (0.5M)</td>
<td>PR-V4231</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>SDS</td>
<td>Reagent</td>
<td>Fisher</td>
<td>BP166-100</td>
<td>&nbsp;</td>
<tr>
<td>Maleic acid</td>
<td>Reagent</td>
<td>Fluka</td>
<td>63189</td>
<td>&nbsp;</td>
<tr>
<td>Boehringer Blocking Reagent</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11096176001</td>
<td>Use at 2% in MABT. To make, heat with MABT for 20 mn at 60C. Can make a stock and store in 1-5 ml aliquots at 20C.</td>
</tr>
<tr>
<td>Heat inactivated sheep serum</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>013-000-121</td>
<td>Dissolve in 10 ml H2O; heat 550C, 30 mn. Aliquot at 1ml, -20C.</td>
</tr>
<tr>
<td>Anti-Digoxigenin-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>NBT/BCIP stock solution</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11681451001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Fluorescein-AP, Fab fragments</td>
<td>Reagent</td>
<td>Roche Applied Sciences</td>
<td>11426338910</td>
<td>&nbsp;</td>
<tr>
<td>2M Tris</td>
<td>BP1759-500</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vector Red Alkaline Phosphatase Substrate Kit</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>SK-5100</td>
<td>&nbsp;</td>
<tr>
<td>1,2,3,4-tetrahydronaphthalene</td>
<td>Reagent</td>
<td>Sigma</td>
<td>429325-100ML</td>
<td>Under hood</td>
</tr>		
</tbody>
</table>
</div>

double whole mount in situ hybridization of early chick embryos

鸡胚是一个有价值的工具，在早期胚胎发育的研究。其透明度，可获得性和易于操纵，使其用于研究基因表达在脑，神经管，体节和心脏原基形成的理想工具。本视频演示了不同的步骤，在2色整装原位杂交;首先，胚胎从卵解剖和多聚甲醛固定。二，预杂交胚胎处理。具有两种不同的探针，再加上挖一个，并再加以FITC标记的杂交胚胎。杂交过夜后，胚胎培养与辛加上抗体。辛基板颜色反应的进行，和感兴趣的区域显示为蓝色。胚胎，然后加上与FITC标记抗体孵育。胚胎处理颜色用FITC反应，和感兴趣的区域显示为红色。最后，胚胎是固定的，为phtograph处理和切片。还介绍了故障排除指南。

Watch this Scientific Journal Video about Double Whole Mount in situ Hybridization of Early Chick Embryos at JoVE.com

Double Whole Mount in situ Hybridization of Early Chick Embryos

本视频演示了原位杂交的方法，其中2个不同的基因的时空表达模式可以在年轻的鸡胚显示2色整装。这种方法最初是由大卫威尔金森，多明戈斯恩里克，菲尔英厄姆和大卫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 ...

double-whole-mount-in-situ-hybridization-of-early-chick-embryos

Research

JoVE Journal

Biology

12.3K 次观看.  Institute of Biosciences and Technology - Texas A&M Health Science Center. 本视频演示了原位杂交的方法，其中2个不同的基因的时空表达模式可以在年轻的鸡胚显示2色整装。这种方法最初是由大卫威尔金森，多明戈斯恩里克，菲尔英厄姆和大卫ISH - Horowicz。

视频: Double Whole Mount in situ Hybridization of Early Chick Embryos

Method for Culture of Early Chick Embryos ex vivo (New Culture)

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  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

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

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 ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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 ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

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 ...

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

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

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

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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 ...