作者希望，在雪城大学的博士和玛丽莲克尔承认自己的角色比较脊椎动物生物学课程管理生物系。艾伯森实验室授予R21DE019223支持健康/国立牙科和颅面研究所研究所的国家机构，以及来自美国国立卫生研究所/国家老化研究院授予R01AG031922。

1。目标基因的质粒转化第一部分：质粒转化 （所需时间：3小时加孵育过夜） <ol><li>暖SOC营养肉汤在42℃水浴（每秒500μL反应） </li><li>在冰上解冻胜任细胞</li><li>加入1μL质粒25μL胜任细胞</li><li>置于冰上20分钟</li><li>在42℃水浴热休克细胞为45秒</li><li>立即置于冰上2分钟的细胞</li><li>每小瓶细胞加入500μL42℃的营养肉汤</li><li>摇37 ° C为2小时每分钟旋转255（RPM） </li><li>板75μL卢里亚Bertani（LB）琼脂平板转型混合</li><li>孵育板倒在37 ° C过夜</li></ol>第二部分：E大肠杆菌文化 （所需时间：15分钟加孵育过夜） <ol><li>对于每一个反应，等分3 mL LB培养基中，并成为一种文化管3μL50 mg / mL的氨苄青霉素</li><li>从琼脂平板刮1菌群，并添加到每个培养管</li><li>摇文化管在37 ° C时，255个RPM过夜</li></ol>第三部分：质粒准备 （所需时间：1.5小时） <ol><li>隔离质粒从过夜培养，使用5首相FastPlasmid试剂盒（产品编号2300000） </li><li>要检查准备质粒的存在，运行与溴化乙锭染色的1％的Tris -醋酸EDTA（TAE）凝胶洗脱试剂盒的DNA </li></ol> 司徒高辛标记的RNA探针合成2。 第一部分：线性质粒 （所需时间：2.5小时） <ol><li>在1.5 ml离心管中，结合： <table border="1"><tbody><tr><td>制备质粒</td><td> 20毫升</td></tr><tr><td>限制性内切酶* </td><td> 2毫升</td></tr><tr><td>缓冲区** </td><td> 10毫升</td></tr><tr><td> 10X牛血清白蛋白（BSA） </td><td> 10毫升</td></tr><tr><td>焦碳酸二乙酯（DEPC）水</td><td> 58毫升</td></tr><tr><td></td><td> 100毫升</td></tr></tbody></table></li><li>在37 ° C 2小时 *根据与质粒 **随限制性内切酶</li></ol>第二部分：转录 （所需时间：3小时） <ol><li>在1.5 ml离心管中，结合： <table border="1"><tbody><tr><td>线性质粒</td><td> 4毫升</td></tr><tr><td> 10X转录缓冲液** </td><td> 4毫升</td></tr><tr><td>地高辛标记组合</td><td> 4毫升</td></tr><tr><td>聚合酶* </td><td> 2毫升</td></tr><tr><td>核糖核酸酶抑制剂</td><td> 2毫升</td></tr><tr><td> DEPC水</td><td> 24毫升</td></tr><tr><td></td><td> 40毫升</td></tr></tbody></table></li><li>在37 ° C孵育1小时</li><li>加入2μL聚合酶* </li><li>在37 ° C孵育1小时</li><li>加入2μL的DNA酶</li><li> 37 ° 20分钟的彗星 *根据与质粒 **与聚合酶变化</li></ol>第三部分：降水 （所需时间：5分钟加2小时孵育过夜，1小时到离心机以及重新挂起） <ol><li>加入4微升0.2M EDTA </li><li>添加5μL4M的氯化锂</li><li>加入150μL冰冷的100％的乙醇</li><li>在-80℃2小时孵育隔夜</li><li>在4 ° C，倒出离上清在30分钟14000转的离心机</li><li>干颗粒为7分钟</li><li>再暂停20μLDEPC水</li><li> 37 ° 5分钟</li></ol>第四部分：分馏 - 只有执行探头尺寸大于0.6 KB （所需时间：根据探头大小而定，一般不超过20分钟） <ol><li>在1.5 ml离心管中，结合： <table border="1"><tbody><tr><td> RNA探针</td><td> 20毫升</td></tr><tr><td> DEPC水</td><td> 12毫升</td></tr><tr><td>碳酸氢钠</td><td> 4毫升</td></tr><tr><td>碳酸钠</td><td> 4毫升</td></tr><tr><td></td><td> 40毫升</td></tr></tbody></table></li><li>在60℃水浴孵育。相应的方程的孵化时间： 时间（分钟）=（KB - 所需KB起）/（KB x 0.11 x开始所需KB） 平均所需的大小= 0.6 KB </li></ol>第五部分：最后降水 （所需时间：5分钟犹特再加上2个小时的孵育过夜，1小时到离心机以及重新挂起，1小时凝胶电泳） <ol><li>加入40μLDEPC水</li><li>新增8μL醋酸钠</li><li>添加1.04μL冰醋酸</li><li>添加240μL，冰冷的100％的乙醇</li><li>在-80℃2小时孵育隔夜</li><li>在4 ° C，倒出离上清在30分钟14000转的离心机</li><li>干颗粒为7分钟</li><li>再暂停20μLDEPC水</li><li>涡旋混合</li><li>要检查的riboprobe存在，用溴化乙锭重悬的解决方案上运行了1％TAE凝胶染色</li></ol> 3。 原位杂交技术在全山第一部分：固定的胚胎和蛋白酶K消化 （时间要求：固定过夜，加上4小时的存储的第二天，2.5小时从存储到第二部分） <ol><li>收集上演斑马鱼的胚胎，并固定在4％多聚甲醛（PFA），4℃过夜° C </li><li>洗净的磷酸盐缓冲液固定胚胎，含有0.1％，每次10分钟吐温20（PBST）的3倍</li><li>为了确保胚胎暴露于实验试剂，手动dechorionate胚胎（如有必要），用细尖镊子</li><li>脱水洗衣机梯度为25％和50％PBST甲醇一小时每次洗的胚胎，然后将其存储在100％的甲醇，在-20 ° C </li><li>准备使用时，补充水分在50％（2次）和25％（1次），每次10分钟PBST甲醇洗涤的胚胎在PBST。最后在100％PBST洗，每次10分钟的2倍。 PBST /甲醇洗涤的确切时间并不重要</li><li>死神胚胎在PBST 10％的氢过氧化物的解决方案，如果有必要，消除黑暗的颜料。胚胎中的过氧化氢溶液中孵育10-20分钟，取决于胚胎的年龄，和离心管帽应保持开放，以防止空气压力建立</li><li>精华与50 mg / mL的蛋白酶K的胚胎在PBST稀释1:5000为3-15分钟，根据胚胎的年龄</li><li>重新修复的胚胎在4％，煤灰30分钟，然后在PBST洗3次，每5分钟</li></ol>第二部分：Riboprobes杂交 （所需时间：3小时加杂交过夜，1.5小时，第二天第三部分） <ol start="9"><li> Prehybridize与预杂交液（小灵通）在70℃水浴预热2-3个小时的胚胎</li><li>取出小灵通，加入0.5毫升的杂交液和1.5μL，以前合成的地高辛标记riboprobes，在70℃过夜孵育。温度内波动取决于几度riboprobe目标</li><li>第二天，洗胚胎在70 ° C的75％，50％，25％和2倍生理盐水，每次10分钟的柠檬酸钠（SSC），小灵通分级的解决方案，然后洗为30分钟，在0.2X SSC在68 °彗星</li><li>洗净，每次10分钟，在室温下马来酸缓冲器（MAB）的2倍</li></ol>第三部分：（α- DIG），抗地高辛抗体孵育 （所需时间：3小时加通宵块，2.5小时，第二天第四部分） <ol start="13"><li>胚胎转移到12孔板</li><li>在封闭液1-2毫升块前胚胎在室温下至少3小时</li><li>同时，前块在此解决方案，准备第二卷的封闭液和稀释的抗地高辛抗体1:2000的抗体</li><li>删除前块，加1-2毫升预先阻止α- DIG解决方案，孵育过夜，在4 ° C </li><li>第二天，在人与生物圈洗的胚胎。允许胚胎孵化在人与生物圈计划第一次为5分钟，然后执行缓冲区的改动和孵化为两个10分钟，30分钟和60分钟的时间间隔。确切的时间是没有必要的的</li><li>洗净胚胎碱性磷酸酶（AP）的缓冲区每5分钟的3倍</li></ol>第四部分：染色和最后的处理 （所需时间：1小时染色过夜，取决于使用riboprobe，可以存储在甘油甘油洗涤，每甘油洗6-10小时，开始4小时） <ol start="19"><li>胚胎染色溶液加入1-2毫升，敷箔板，并检查定期（约每20分钟一班）染色，直到染色就足够了</li><li>用PBST 2次，每次5分钟洗净胚胎停止染色反应</li><li> 10分钟的清洗（1次）在25％和50％（2次）PBST甲醇，然后用100％甲醇脱水胚胎，以消除背景染色</li><li>允许胚胎孵化在室温下至少2个小时，在100％甲醇</li><li>补充水分在使用10分钟的洗涤，在50％（2次）和25％（1次）甲醇在PBST PBST，然后洗净PBST 2次100％ </li><li>转移染色Ëmbryos PBST在80％的甘油溶液，使用的清洗和存储的分级系列，4 ° C。 </li></ol>食谱： <ul><li> LB琼脂平板上，10克的LB琼脂+ 250 mL蒸馏水中，高压灭菌，冷却到触摸时添加250μL，氨苄青霉素，每个培养皿倒入15-20毫升温暖的解决方案，使琼脂凝固</li><li> LB肉汤12.5克LB肉汤200毫升蒸馏水中，高压灭菌器，让冷却后再使用</li><li> 1％TAE凝胶，40毫升1X TAE 2μL，溴化乙锭0.4克琼脂糖;负载7μL的质粒（质粒转化目标基因）或3μLriboprobe和4μLDEPC水（ 原位 DIG标记的RNA探针合成） + 1μL上样染料，5μLDNA梯状</li><li>预杂交解决方案的50％甲酰胺，5X SSC，9.2mM柠檬酸，1％Tween - 20的</li><li>杂交液，预杂交液加500μg/ mL的tRNA和50微克/ ml肝素</li><li>马来酸人与生物圈- 100MM，150MM氯化钠，0.2M氢氧化钠，0.1％Tween - 20的pH值至7.5 </li><li>封闭液3部分人与生物圈计划，第1部分的10％勃林格阻断试剂，在人与生物圈计划，第1部分热量停用羔羊血清</li><li> AP缓冲60MM的Tris - HCl pH值至9.5，氯化钠60MM，30MM MgCl 2的 ，0.1％Tween - 20的</li><li>染色液5 - 溴4 - 氯-3 - 吲哚磷酸（BCIP）和硝基蓝四氮唑（NBT）在AP的缓冲区</li></ul> 4。代表性的成果正确执行时，NBT，BCIP和碱性磷酸酶之间的反应将形成一个紫色的沉淀，应该对斑马鱼的胚胎出现一个紫色的污点。 Riboprobes应以前合成的cDNA对应感兴趣的基因。因此，可以断定任何污点的可视化表示感兴趣的基因的斑马鱼已经在那个特定的发展阶段的转录。对于这门课程的目的，riboprobes合成aldh1a2（以前raldh2; Begemann 等，2001;图1）; fgf8a（Reifers等 ，1998; 图 2）; deltaC（奥茨等人，2005年）; myod1（温伯格等人，1996年）; shha（克劳斯等人 ，1993年），pax2a（品牌等，1996;图3）和myl7（以前cmlc2。Yelon等 ，1999）的cDNA。染色预计在中线和包括体节，tailbud，肌节，脑，和心脏的解剖结构。Ace/fgf8a突变预计将有许多这些结构上的缺陷。染色轻松地可视化，使用标准的解剖显微镜。其他来源包含更多的信息，对拟到这里描述的类似的技术和故障排除（克拉克，2003年，德科斯塔等人，2009; Schmoldt 等 ，2009; Schoenwolf 2009年）。 <img src="/files/ftp_upload/2533/2533fig1.jpg" alt="图1" /> 图1：斑马鱼的胚胎24小时受精后，已与特定riboprobes为aldh1a2杂交。特殊染色可以看出眼睛，后脑，胸鳍芽原基和体节。前路是到顶部，后是到了谷底。 <img src="/files/ftp_upload/2533/2533fig2.jpg" alt="图2" /> 图2：在13体节期的发展已为 fgf8a探头具体杂交斑马鱼的胚胎。特殊染色可见在端脑，间脑背侧，中脑，后脑边界，体节，和tailbud。腹是向左，背是正确的。 <img src="/files/ftp_upload/2533/2533fig3.jpg" alt="图3" /> 图3一个22小时受精后斑马鱼的胚胎已与pax2a，一个强大的标志，有助于神经系统的可视化riboprobe具体杂交。可以看出，在脉络膜裂，中脑，后脑边界，奥迪囊泡，和脊髓神经元特异性染色。背视图与左前方。

<ol>
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J., Riggleman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+zebrafish+MyoD+in+wild-type,+no+tail+and+spadetail+embryos.">Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos.</a> Development. 122, 271-280 (1996).</li><li>Yelon, D., Horne, S. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+expression+of+cardiac+myosin+genes+reveals+regulated+aspects+of+heart+tube+assembly+in+zebrafish.">Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.</a> Dev. Biol. 214, 23-37 (1999).</li></ol>

愿望是在一个比较脊椎动物生物学实验室课程，帮助学生理解遗传学解剖发展中的作用，通过已知的基因表达模式的可视化。学生对于课程的第一部分，代表了几个不同的脊索动物类群微生物进行解剖，让他们充分的时间来学习，理解，比较，对比脊椎动物解剖。 课程的第二部分的介绍，学生分别获得了正式的演讲，描述斑马鱼的发展和解剖。希望实验的方法和预期结果进行了讨论。然后给学生们在somitogenesis和6个阶段发展的prim的活的斑马鱼，并在2和第5天受精后（DPF），在解剖显微镜下检查。这是给学生更好地了解了斑马鱼的胚胎是什么样子，发生过个体发育的形态学变化的类型。 在未来的实验室会议，学生们希望在其中先前已进行斑马鱼的胚胎。他们被要求为每个感兴趣的基因（riboprobe使用）的基因表达模式的研究和描述。拟使用的胚胎是来自杂合 ace/fgf8a线的成员之间的交配。根据孟德尔遗传，25％的胚胎从ace/fgf8a交配，预计可纯合突变体，并表现出许多集中在这门课程的解剖结构的缺陷。在艾伯森实验室发表的报告和未发表意见的基础上，预计在大脑和心脏循环不当的缺陷，以及在体节上的缺陷（品牌等 ，1996;。艾伯森和Yelick，2005年个人意见）。 学生被要求检查所有标本，野生型（杂合子动物是在发展的初期阶段从野生型的兄弟姐妹没有区别）和纯合突变体，对提出的每一个基因的表达模式。然后，他们被要求写出实验报告，描述其结果，并根据他们的解剖学和遗传学，有缺陷的基因的表达如何可能有沉淀解剖畸形知识。 学生们似乎接受这种兴奋和好奇的实验室工作。大多数从来没有使用拟之前和这部分的课程非常感兴趣。学生发现有趣的斑马鱼胚胎中的基因表达不同的图案，有的甚至描述的染色模式的可视化关联，他们与著名的设计和符号，如笑脸，。造成实验室的报告显示，学生希望协议在具体的解剖结构基因的表达了一个大致的了解。同学们也需要了解该基因的具体功能研究过程中使用的实验室（斯蒂克尼等人 ，2000年希望; Huelsken 等，2002;。Geetha - Loganathan 等，2008A;。Geetha - Loganathan 等 ，2008B ）。很明显，但是，某些学生的信号通路有限的知识背景和利益的基因。在对这些概念的更多信息，想介绍性的讲座可能是一个值得欢迎的除了希望在未来比较脊椎动物生物学课程。 由于协议一般需要连续四天，根据课程计划，学生可能只能够完成的实验在课堂上，教师必须负责其余部分。在我们的比较脊椎动物生物课，学生在实验室完成的染色反应，而教学助理执行前面的步骤。 ，如果它是首选有执行希望在课堂上的学生，该协议可以在几个实验室会议，根据类的时限亚基可分为。如果是不可行的学生完成整个协议，因为它是在这里，由于一个星期只有一次实验室会议，学生可以在课的开始添加染色溶液，取决于所使用的riboprobe，染色完成在一个小时内。污渍发展所需的时间差别很大每个riboprobe和各种实验条件，并应在课前预先确定的。值得注意的是，如果学生只会在实验室开发的污渍，教官将负责所有前面的步骤，这将需要大量的时间和努力，在课堂以外的。如果需要，可以参考书较短的替代免疫组织化学，使用抗体蛋白定位可视化的标签，但是在这个时候，斑马鱼发育基因特异性抗体不容易获得。另一种选择是，执行不同的脊椎动物物种的愿望一个ND学生比较相同的基因表达模式，在不同的生物体（Pizard等，2004;荒等，2007;新兴的模式生物，2008年，新兴的模式生物，2010） 。 利用的总体目标，希望在一个比较脊椎动物生物学课程学生演示如何使用分子生物学技术研究解剖发展。它还提供了一个机会让学生猜测如何改变基因的表达可能不仅导致发育畸形，但也进化改变。进化发育生物学（通常称为“演化DEVO”）正式的，快速增长的这一研究领域，旨在通过发展联系起来，基因型和表型，并阐明进化变化的潜在机械基地。随着这一领域的崛起，更多的生态学家，有机体生物学家和生理学家采用分子生物学技术，在他们的研究。我们抗衡，希望在一个比较脊椎动物生物学课程的使用将有助于保持课程的日期与目前的技术和概念的研究进展，并结合生物子域，以方便更好上层生物学课程的水平对齐。此外，这个一体化的方法，将提供学生有机会学习一门课程的生物研究技术的分类，导致一个更多元化的教育和促进未来跨学科的科学研究。

<ul><li>高压灭菌器</li><li>微型离心机</li><li> 37℃培养箱（与振动筛） </li><li> 4 ° C微型离心机</li><li>水浴</li><li>涡旋</li><li>凝胶电泳仪</li><li> -20 ° C冰箱</li><li> -80 ° C冰箱</li><li>摇杆/摇床</li><li> 4 °彗星冰箱</li><li>解剖显微镜（最好的教学屏幕或相机） </li></ul>

using whole mount in situ hybridization to link molecular and organismal biology

整装原位杂交（拟）是一种在分子生物学实验室，用于研究基因的表达，通过特定的mRNA转录的整个装入标本内的本地化的常用技术。高层本科比较脊椎动物生物学实验室在美国雪城大学课堂上使用这种技术（从2005年艾伯森和Yelick，改编）。前两个三分之二的比较脊椎动物生物学实验课程给学生学习的机会，代表各种脊索动物类群，主要是通过传统的解剖和使用模式的几种有机体的胚胎学和大体解剖。参与课程的最后部分教学通过解剖观察脊椎动物聘用那些希望对斑马鱼的胚胎分子生物学技术的发展创新的方法。杂合成纤维细胞生长因子8（fgf8a）突变系，ACE，使用。由于孟德尔遗传，ACE intercrosses生产的野生型，杂合子，纯合子 ace/fgf8a在1:2:1的比例的突变体。 RNA探针与已知的表达模式在中线和发展，如心，体节，tailbud，肌节，和脑的解剖结构。愿望是与学生在课堂上表演的染色反应，使用斑马鱼在13体节和拘谨6个阶段进行。斑马鱼的胚胎处于不同发展阶段的研究，使学生的能力，以观察这些解剖结构的个体发生改变。此外，一些ace/fgf8a突变体显示不当的心脏循环和体节和大脑发育的缺陷。学生在这个实验室观察使用外部解剖以及基因表达模式的各个器官系统的正常发展。他们还发现和描述显示不当解剖的发展和基因表达（即假定的突变体）的胚胎。 机构已经不拥有必要的设备，或者是有限的资金用于实验室和课外创新的导师，试剂和仪器的财务成本可能是一个需要考虑的因素，会的时间和精力的一部分无论设定的导师。不过，我们抗衡，希望在使用这种类型的课堂教学实验室设置，可以提供一个发育遗传学和解剖学之间重要环节。随着技术的进步，并能够在分子水平上研究有机体的发展变得更容易，更便宜，越来越受欢迎，许多进化生物学家，生态学家和生理学家在分子生物学领域的研究策略。使用一个比较脊椎动物生物学实验室课堂希望是如何分子和解剖可以在一个单一的课程衔接的一个例子。这使上层大学生现代生物学研究技术，导致一个更多元化的教育及促进未来的跨学科的科研实践的机会。

Watch this Scientific Journal Video about Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology at JoVE.com

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

整装<em&gt;原位</em&gt;杂交（拟）是用于高层本科比较脊椎动物生物学除了脊椎动物解剖课程。这使学生有机会来研究基因表达模式以及大体解剖，连接分子和有机体生物学研究的一门课程内。

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 ...

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JoVE Journal

Biology

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

21.5K 次观看.  Syracuse University. 整装原位杂交（拟）是用于高层本科比较脊椎动物生物学除了脊椎动物解剖课程。这使学生有机会来研究基因表达模式以及大体解剖，连接分子和有机体生物学研究的一门课程内。

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

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

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 ...

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 ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

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 ...

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 ...

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 ...