这项工作部分由下列各项资金，以支持RAW：美国国立卫生研究院资助K01 DK083512，DP2 OD008470，和R01 DK100237;三月优生优育基金会罗勒奥康纳入门学者补助金＃5-FY12-75;从科学与生物科学系的圣母院书院大学启动资金。我们特别要感谢伊丽莎白和迈克尔·加拉格尔，连同整个加拉格尔家庭，谁给予的慷慨礼物圣母院大学培养干细胞研究。资助者在研究设计，数据收集和分析，发布的决定，或准备稿子没有任何作用。我们感谢生物科学部的工作人员的支持，和中心斑马鱼研究在巴黎圣母院为他们在我们的斑马鱼殖民地的照顾和福利的杰出贡献。最后，我们感谢我们的研究实验室的成员的意见，讨论和对这项工作的见解，如以及金盏花（Maripooka）和百日草温格特提供自然脱落的猫科动物胡须，使我们的研究最优秀的睫毛工具。

该程序在本协议中所述的斑马鱼胚胎的工作被批准的机构动物护理和使用委员会在Notre Dame大学。注意：在部件1-5中描述的程序被用来生成在有代表性的结果在这里提供的胚胎的图像。在零件1-5描述的过程可以被取代的期望与替代WISH程序12-16或如通过使用特异性抗体特异性蛋白免疫组化标记等可视化技术。在进行平片上WISH斑马鱼胚胎在1-5部分描述的协议，最初的胚胎固定和最后的染色固定的步骤是影响从样品中除去卵黄颗粒的平面安装的能力的主要操作。最好的平面安装的结果时，与刚解冻冰冷的4％多聚甲醛（PFA）/ 1X PBS和固定执行的初始固定获得与冰冷的4％PFA/1x的PBS（这并不需要新鲜解冻），并且它是表示，读者当替代与6-8部分替代组合染色方法考虑这个最后的愿望染色反应。 1，胚胎收集，固定和储存<ol><li>通过将成年雄性斑马鱼（次），并在系统中水的腔室女（次），使它们通过一个分割器分离过夜准备交配笼中。 </li><li>取出分隔成年人之间，并使用内〜交配15-30分钟的细丝网滤茶器来收集具有同等萌芽阶段离合器收集受精卵。 </li><li>转动过滤器上下颠倒并冲洗表面与E3的细流从喷射瓶的胚转移到含有大约25毫升的E3溶液孵化菜分配。 </li><li>收集的魔掌后，将它们分为几盘，使得约50-60胚胎培养在25毫升的E3解每道菜。注：胚胎过度拥挤会导致剧烈的发育不同步内离合器和应注意避免这种情况，以便能够及时收集所需级（S）的。 </li><li>孵化的胚胎在28.5℃下根据斑马鱼标准分段系列11，以使发育评估。 </li><li>用塑料移液管轻轻地放置胚胎的期望数量在选定的时间点，上升到20个体节阶段（19 HPF），入平底微量离心管中。注：请注意在处理幼胚的，因为他们是脆弱的，可以通过挤压或积极处理，传输移液器很容易损坏。可选地，玻璃小瓶，可用于固定的胚胎和处理。对于所有后续协议的洗涤塑料移液管转移可以利用，除核糖体探针的添加和删除（步骤3.11，3.14）。 </li><li>更换E3与冰冷的4％PFA/1x PBS中，固定在其绒毛膜胚胎在4％PFA/1x PBS中4小时，在室温或4℃过夜。提醒：PFA是有毒和PFA的解决方案应在化学罩来处理，而穿着合适的个人防护装备，包括手套，实验室外套。此外，煤灰粉应该用特殊的照顾使原液处理的时候，因为即使是颗粒状的煤灰可以趋于分散，通过静电。典型的PFA是通过加热的1×PBS至沸，以溶解PFA粉末制备的，然后将所得溶液于-20℃下以等分试样储存如果细沉积物中存在的4％的PBS PFA/1x一旦将溶液冷却，过滤灭菌冷却后的溶液通过0.45μm的过滤器系统，其传递等分用于冷冻保存前。只有新鲜制备的或刚解冻的PFA是用于此初始（ 即主要的）胚胎的固定。 </li><li>取出修复和1X PBST洗两次胚胎，然后转移到孵化菜。 </li><li>查看胚胎在立体显微镜下，用两对细镊子，以消除周围的胚胎，这样一对用来轻轻地利用胚胎，而第二对是用来撕开绒毛膜的绒毛膜，。 </li></ol> 2，胚胎Permeablization <ol><li>传输dechorionated胚胎放回微量离心管或玻璃小瓶中，然后用1×PBST冲洗两次，以除去任何残留的绒毛膜碎屑。 </li><li>除去1X PBST和用100％甲醇（MeOH）纯化两次洗涤胚胎。 </li><li>将胚胎在-20℃至少20分钟。注意：胚胎可以存储在-20℃下在MeOH为一年或更长时间。甲醇使绒毛膜粘，因此不进行到这一步，除非绒毛膜已被删除。 </li><li>通过除去100％MeOH中再水化的胚胎和在50％MeOH/1x PBST，30％MeOH/1x PBST用1×PBST在室温下把它们洗干净，每次5分钟，然后两次。&lt;/ LI&gt; <li>加入25微升的刚解冻的蛋白酶K的股票（10毫克/毫升）至50毫升的1X PBST中准备一个新鲜的蛋白酶K工作液（5微克/毫升）。 </li><li>从胚胎中取出1X PBST，然后用蛋白酶K更换工作液孵育基于胚胎阶段：&lt;= 5个体节，持续1分钟; 10-12个体节为1.5分钟; 15个体节2分钟，和20体节3分钟。 </li><li>除去蛋白酶K工作溶液，并用1×PBST洗两次胚胎。 </li><li>除去1X PBST和用冰冷的4％PFA/1x PBS替换为至少20分钟，在室温下进行。 </li></ol> 3，核糖体探针的合成，预杂交，杂交和探针移除<ol><li>组装的反义核糖核酸探针的体外转录在室温下在1.5ml微离心管中反应，同时保持酶在冰上，用含有序列对应于感兴趣的基因（或100-200毫微PCR的DNA模板合成产品或1.5微克线性化的DNA质粒，所述制备12,13），2微升洋地黄毒苷或荧光素标记的核糖核苷酸，2微升合适的RNA聚合酶（SP6，T3，T7取决于其序列上的被掺入到PCR产物或存在于DNA质粒上），2微升的10×转录缓冲液，0.5μl的RNase抑制剂，并带来对20微升的总体积与分子级蒸馏水（DNA酶，RNA酶）。注：10×转录缓冲液应通过将管在37℃水浴10〜20分钟进行预热，并随后涡旋，以确保所有组件都在溶液中。如果白色片状物存在，再次孵化管另一5-10分钟和旋涡。转录反应可以失败或具有较差的产率，如果转录缓冲液是不完全溶解和充分混合。 </li><li>混合组分和孵育在37℃，每个反应2小时在水浴。 </li><li>除去反应TU是（S）从水浴中，并通过加入5微升10×DNA酶I缓冲液，2微升DNA酶I酶和23微升的分子级纯蒸馏水，使总体积为50μl的各破坏样品中的DNA模板，然后孵育各管在37℃，20分钟水浴中。 </li><li>通过加入5微升3M乙酸钠，pH 5.2和110微升冰冷的100％乙醇，然后加入1微升糖原的库存，以促进RNA沉淀可视化在后续步骤中，然后孵育进行乙醇沉淀在各离心管中的管在-20℃下放置至少1小时或过夜。 </li><li>以最大速度离心4℃20分钟，每个离心管中，然后移除100％的乙醇上清液，并替换为100μl的70％冰冷的乙醇。 </li><li>离心机在4℃，每次5分钟离心管中，然后取出70％的乙醇上清液，空气干燥沉淀5分钟，最后加入100μl莫lecular级蒸馏水悬浮颗粒和使用N​​anoDrop分光光度计量化的riboprobe股票浓度。注意：一旦粒料已经进入溶液中，核糖核酸探针库存应保持在冰上，并存储于-20℃。 </li><li>要准备固定胚胎预杂交过程中，从每个样品取出4％PFA/1x PBS中，用1X PBST洗两次胚胎。 </li><li> 25-40胚胎在1x PBST之间传输到每一个单一的平底微量离心管中。注：胚胎过度拥挤会导致染色不均以后，并应注意限制样本量在每管约40胚胎。 </li><li>用500-1000微升HYB +的取代1X PBST每管。 </li><li>放置胚胎的管（S）放入烘箱中在70℃下，孵育它们在该温度下搅拌4小时进行预杂交。 </li><li>通过增加100-500纳克的核糖体探针（地高辛和/或fluoresc准备的riboprobe / HYB +解决方案EIN-标记的），以500微升新鲜HYB +，然后孵育该溶液在70℃下进行到步骤3.11至prewarm它之前至少20分钟。注意：为了检测多种基因的转录，使用单和/或双比色标记，每个对应于这些基因的核糖核酸探针的需要被组合成一个核糖核酸探针/ HYB +鸡尾酒。 </li><li>取出预杂交HYB +从胚胎各管，并更换有足够的riboprobe / HYB +覆盖胚胎。注：通常情况下，200-250微升的riboprobe / HYB +是必要的，以支付在一个平底微量离心管的胚胎。典型地用移液管和阻挡提示，以防止样品间的交叉污染核糖核酸探针/ HYB +添加和删除。 </li><li>孵化的胚胎在70℃下过夜ribroprobe / HYB +。 </li><li>准备清洗解决方案和预温暖他们的下面的一组过夜，在70°C：50％formamide/2x SSCT，SSCT 2倍，0.2倍和SSCT。注：为方便起见，准备50毫升人每个iquots洗涤溶液，因为过量的可在室温下保存，并随后加热以供将来使用。 </li><li>拆下的riboprobe / HYB +使用吸管和障碍的技巧和储存在-20°C。注意：通常每核糖核酸探针/ HYB +混合物可以在没有信号的缩减多次使用（ 例如 ，3个或更多）。虽然重复使用的riboprobe / HYB +与较低的背景有关，但是，请记住，再用核酸探针混合物可能会受到降解。 </li><li>在70℃下用50％formamide/2x SSCT 30分钟洗两次胚胎</li><li>在70℃用2×SSCT 15分钟，洗一次的胚胎</li><li>在70℃下用0.2×SSCT 30分钟洗两次胚胎</li><li>取出0.2×SSCT和在室温下用MABT两次洗涤胚胎。 </li></ol> 4，抗地高辛抗体孵育和检测<ol><li>准备新鲜的块解决方案。 </li><li>取出MABT，换上块解决方案化。 </li><li>在孵育2-4小时，在室温下块解决方案的胚胎。注：较长块孵化提供最佳的结果与年轻的斑马鱼胚胎阶段（&lt;15体节）。过夜执行冗长的程序块中，在室温或室温的组合执行块孵育8-12小时加（最多〜8小时），随后4℃孵育。 </li><li>通过使一个5000倍稀释的新鲜块解决方案（ 例如 ，每5ml块1微升）准备抗洋地黄毒苷抗体溶液。 </li><li>取出块，并添加抗体/块的解决方案。 </li><li>覆盖在箔样品孵育​​过夜，在4℃或4小时在室温下。 </li><li>除去anti-digoxygenin/block与MABT洗胚胎的至少10倍。注：胚胎转移到12孔板的MABT洗。这增加的速度，胚胎可水洗，处理大样本量时，这是有用的，一ND可以很容易地监测染色反应（步骤4.11）在立体显微镜下的进度。注意：在此步骤中的胚胎可以“超洗涤&#39;按前进至步骤4.8，这对于往往给高背景或污渍，需要冗长的监视过程中探针的有用治疗之前，在4℃过夜孵育它们MABT上班的日子。 </li><li>制备预染色缓冲液和所需的染色溶液（紫色或红色的底物）。 </li><li>除去MABT和在预染色缓冲液洗涤胚胎5分钟，在室温下进行。 </li><li>除去预染色缓冲液并加入染色液。 </li><li>通过检查胚胎的立体显微镜下的外观监察室温染色反应的进程每隔15-30分钟。注意：在染色反应的速率可以通过将所述样品4℃，如果需要，得到更多的时间用于染色完毕后进行减速。一旦冷却至4℃，但是，该反应不能被温热至室温加速。 </li><li>当染色反应完成后，去除染色液并更换用1×PBST。 </li><li>用1×PBST 5分钟，洗两次。注意：如果没有检测到其它基因与另一基板，然后固定在冰冷的4％PFA/1x PBS样品至少20分钟，然后继续执行步骤6.1。否则，继续执行步骤5.1。 </li></ol> 5，抗荧光素抗体孵育和检测<ol><li>取出1X PBST和用0.1M甘氨酸，pH值2.2的两倍洗胚胎15每在室温下最小。注意：该时间为甘氨酸清洗是必不可少的，以完全关闭所述第一染色反应。 </li><li>除去0.1M甘氨酸和与MABT两次洗涤胚胎5分钟，在室温下进行。 </li><li>通过使一个5000倍稀释的新鲜块解决方案（ 例如 ，每5ml块1微升）准备抗荧光素抗体溶液。 </li><li>取出MABT并添加抗体/块的解决方案。按照步骤4.6-4.13进行荧光标记ribroprobe的检测。注意：当最终的染色反应完成后，至关重要的是，样品的最后固定在冰冷的4％PFA/1X PBS中完成。固定用温暖的PFA溶液趋向于用粘蛋黄是很难从胚胎解剖和deyolking为平坦安装过程中去除相关联。 </li></ol> 6，胚胎解剖及胚胎的初始Deyolking <ol><li>选择一个固定，染色胚胎的平面安装。 </li><li>使用移液管，以所选择的胚胎的样品放置到含有1x PBST中的塑料或玻璃培养皿。 </li><li>将培养皿在立体显微镜下，并用一对细镊子或睫毛的工具，使得一个侧视图，得到与头部和尾部端可视滚动胚胎。 </li><li>使用一对细镊子，使在日的切口ë蛋黄在位于中央的头部和胚胎的尾部之间的位置上，同时用第二对细钳子保持胚胎，以便提供杠杆作用为蛋黄切口。注意：可替换地，使两个主要的切口中的蛋黄，1接近头部和其他临近的胚胎的尾部。 </li><li>用细镊子和/或睫毛工具舀出蛋黄从卵黄细胞腔。 </li><li>轻轻冲洗胚胎与1X PBST去除杂散蛋黄。 </li></ol> 7，精细Deyolking胚胎<ol><li>胚胎转移到一个平面载玻片用1-2滴1X PBST的。 </li><li>使用睫毛工具和细镊子与腹侧（蛋黄）的一面朝上于载玻片上轻轻放置胚胎。 </li><li>胚胎拖动到使用睫毛工具1X PBST溶液的边缘，这样的胚胎仍然贴在玻璃上滑动的平面位置。 </li><li>轻轻刮掉使用睫毛胚胎的腹面醇以除去卵黄颗粒无翻录的胚胎。同时用一双细镊子的锚胚胎同时与睫毛工具刮。注：去除蛋黄颗粒可能需要反复刮5-10分钟。 </li><li>如果PBST溶液变得混乱与过量的蛋黄或开始蒸发，用的是塑料或玻璃移液管加入1-2滴新鲜的1X PBST向滑动，然后将胚胎拖到面积为进一步卵黄颗粒的清洁溶液去除。 </li><li>时蛋黄已经令人满意地除去，用移液管轻轻地移动胚胎放回含有1x PBST中的最后冲洗，以除去杂散卵黄颗粒的塑料或玻璃培养皿。 </li><li>使用塑料或玻璃移液管转移到含有100％的甘油一塑料或玻璃培养皿转移deyolked胚胎。 </li><li>孵化胚胎在100％甘油〜5分钟，以适应胚胎。 </li></ol>8，安装在载玻片上<ol><li>该deyolked胚胎移植到1-2滴100％的甘油干净的载玻片上。 </li><li>与腹侧（蛋黄）朝向玻璃滑动位置的胚胎。 </li><li>使用睫毛工具，拖动胚胎甘油下降的边缘使胚胎仍然贴在玻璃上滑动的平面位置。如果胚胎轴弯曲，用细镊子轻轻地做很小的切口，在剩下的蛋黄细胞膜，以舒缓紧张情绪，使胚胎能平放在同一条直线轴的幻灯片。 </li><li>将橡皮泥的小草皮上的四个角落的18×18毫米的玻璃盖玻片。注意：作为一种替代方法，小滴，真空润滑脂15可以取代橡皮泥。 </li><li>放置在玻璃盖玻片上缓慢的胚胎，钓鱼盖玻片，以尽量减少在样品周围的甘油气泡的产生。 </li><li>加100％的甘油的附加压降在玻璃盖玻片一侧填盖玻片与载玻片之间的空间。 </li><li>按下缓慢而仔细地四个角上盖玻片牢牢定位在玻璃之间的胚胎和消除漂流。注意：小心不要挤压胚胎。 </li><li>如果胚胎是不是定位为需要，用细镊子取出盖玻片。使用睫毛工具重新定位胚胎和/或细镊子进行额外的切口，使胚胎可以重新定位。重复步骤8.4-8.7。 </li><li>观察和/或图像使用一个立体声或复式显微镜标本。 </li><li>存储扁安装准备与平板在黑暗中一个纸板滑动支架在4℃数周或暂时在室温下。注意：WISH污点会褪色逐步随着时间的推移，特别是红色衬底的反应，并且不能在甘油无限期地保留下来。紫渍也淡出更容易，如果保存在甘油相比，PFA（参见步骤8.11）。有趣的是，已经发表了固定的胚胎中permount而不是丙三醇可在室温下15使无限期储存。 </li><li>对于长期储存的紫色染色基板样品，取出盖玻片，并冲洗在1x PBST中冲洗两次，胚胎，然后转移到一个离心管中，用4％的PBS PFA/1x用于长期存储。注：另外，在1x PBST胚胎可用于补充分析，如DNA分离的基因分型进行处理。 </li></ol>

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作者什么都没有透露。

斑马鱼已被证明是近年来整个研究界一个极有价值的模式生物。斑马鱼表现出遗传保护相当程度与高等脊椎动物，以及有关他们的解剖结构，繁殖和生命周期优势使得这个物种非常适合进行实验分析。此外，适用于斑马鱼的分子生物学技术的进步，进一步推广使用在科学研究这一模式生物。例如，已经产生了大量的各种转基因斑马鱼线来研究疾病的进展，并回答背后发展的关键机制，包括器官的许多根本性的问题。 因此，根据在这两个疾病和发育的研究动态使用斑马鱼，细胞标记方法和信号量化为数据分析的重要方面。而采用荧光的方法tibodies可以用来检测蛋白表达的转基因斑马鱼中，研究人员通常依靠像WISH 12-16标准程序，以检查基因转录物在一个固定的，非转基因斑马鱼样品的时空定位。事实上，愿望是在生物学16使用最广泛的技术之一，是用来描述基因突变和化学基因扰动的表型的重要工具。然而，愿望是与一些潜在的隐患和挑战有关。为了证实该反义ribroprobe的特异性，义的核糖核酸探针可以被用作一个控制，以评估的反义核糖核酸探针染色模式的特异性。而第4和第5的标记和检测地高辛标记的探针其次是荧光素标记探针的顺序呈现，这个顺序可以颠倒。通常情况下，较弱的信号（与基因表达水平较低）最好与地高辛标记的探针和这个污点检测用紫色基底，随后检测与使用红色衬底更强的信号（更丰富地表达的基因）的染色开发的第一个。然而，如果两色反应将要进行的，因此建议标签和基片的各种组合进行测试，以发现最适合于数据采集和分析的过程。此外，所提供的时间封锁，在4-5节是专为胚胎年龄小于24小时受精后提供抗体孵育和洗涤;漫长的间隔与旧的胚胎这些相同的步骤可能会导致在样品盐分积累。用于故障排除的标准斑马鱼胚胎WISH广泛的技巧已经记载在文献12-16。可以说是最常见的困境是高背景。我们建议，如果需要增加MABT清洗的步骤4.7至15-20洗涤次数，不过，在我们的经验中除了隔夜MABT“超级洗”的给出了优异成绩进入步骤4.8前10名或以上短MABT清洗一起使用时。另一个例子困境差探头渗透，它可以发生与漫长的核糖核酸探针。剪切的riboprobe以下步骤3.6通过碱性水解可以抵消这一点。在我们年轻的样本经验，剪切探头通常不必需的。然而，人们应该记住，参数这样可以在WISH实验的结果是导致因素，我们建议检查已经在社会上公开提供根据需要细化自己的实验参数，希望对这些有用的故障排除说明研究12。 除了 ​​传统的WISH技术12-16，出现了在WISH的方法和已制定的备用贷款协议的不断出现。这些包括信号检测的新方法，如通过使用荧光的cence 17-19，给方法，使小分子RNA 20的定位。即使如此，尽管已对当前小区标识的方法的许多改进，这些程序的有效性被否定，如果污渍（次）不能轻易感兴趣的样品中在期望的时间点显示。这种限制是有问题的研究者，特别是当它涉及到斑马鱼个体发育的早期胚胎阶段，这有可能导致差距在我们这出现在这些时间不同发育过程的理解。在正常的斑马鱼发育，卵黄囊将逐步在尺寸减小的胚胎年龄11。因此，在这些后来的发育阶段（&gt; 24小时受精后），WISH染色是相当简单的分析，因为胚胎较大相比，其毗邻的卵黄囊。然而，这不是从尾芽期的情况下，以早期体节形成（当胚胎质量被定位围绕蛋黄），其中所述不透明卵黄囊有时可以掩盖染色的可视化。因此，平面安装的方法被设计来帮助减轻与早期的斑马鱼胚胎的样品（图式于图1和图2）的特征相关联的成像和数据分析的问题，并从此基因突变孤立的系统的表型被广泛地用于从第一次大规模遗传筛选21。 由于平面安装技术的出现，早期发育阶段的分析已经显著提高。一旦蛋黄是从胚胎取出，因此能够打好胚胎平面上的成像所需的方向载玻片。此二维位置能够观察整个胚胎的一次，这消除了需要旋转样本。许多组织都位于胚胎的广阔领域，如形成血液和肾脏的前体。此外，一个可能需要在同一时间，以比较几种不同的开发组织。平面安装使研究者能够同时查看这些细胞群的整个域，从而可以极大地帮助量化，如面积测量的组织或细胞计数。这种技术已最终帮助导致有关的各种相关造血，神经，器官和重要的发展机制的许多发现。因此，一旦掌握了，对于这个简单的技术研究人员的应用已相当可观。 例如，在一项研究造血过程中检查谱系选择，平板装载斑马鱼胚胎的制剂在14和18体节阶段（β）用于说明发生在之间的PU.1锌指转录因子的表达模式的变化野生型和GATA1啉注射的胚胎22。这种方法使一个扩大PU.1结构域的18 SS的检测，表明GATA1是最有可能调节PU.1的表达在中间细胞团（ICM）围绕该时间点。染色不同红细胞基因，包括gtpbp1和底物蛋白的其他平面安装的胚胎表明这些基因的表达的损失在VLT突变体，分别为GATA1 - / - 。进一步的分析显示，erythoid基因像biklf和被称为是独立的转录因子GATA活动testhymin的表情没有任何变化。因此，在与他们的其他实验结果的同时，它显露GATA1是在调节细胞的骨髓，红细胞系中的分化是必不可少的。神经嵴发育的研究中，平片被用来研究crestin表达的勃朗峰（MOB M610）在一项研究中突变体检查勃朗峰和tfap2a基因功能23的角色。在10和20的ss，crestin表达完全废除的头部和下降的暴徒M610突变体主干区域相比，正常野生型表达，最终帮助本组总结的关于勃朗峰的重要性和tfap2a调控在神经嵴形成。此外，在器官而言，平片已启用不同的结构域的存在，在早期肾脏祖字段中发现的斑马鱼胚胎肾脏的发育过程中，被称为鱼头。在斑马鱼胚胎提供一个保守的，但在解剖学直截了当的系统来研究肾祖细胞如何构成的鱼头肾单位功能单元产生，被称为nephrogenesis 6,7的处理（ 图4）。平板安装分析相当有用的重域文件NAL祖细胞，并在鱼头图案6,7（ 图5），这是前所未知的解剖变化RA信号的结果。总而言之，这些例子表明，确实有这平坦的安装协议的正常发育和疾病状态过程中发生的不同过程的研究很多广阔的应用前景。 从概念上讲，这款平板安装过程相当简单的整体。然而，技巧的掌握这种方法所需的操作和程度可能相当具有挑战性的掌握在没有视觉演示。因此，该协议被编译，使研究人员，特别是那些新来的斑马鱼模型，有机会更好地了解如何执行这项技术，并分享我们在这可以优化组织处理试剂的意见。我们希望这一协议最终将允许其他人在社会上完善最理想的样本条件为uSED对优质成像，数据分析，和他们在出版物结果沟通。最终，这应该使研究人员能够克服斑马鱼的解剖学障碍在早期胚胎发育，这可能掩盖了实验结果的介绍，并通​​过使用这个简单而显著技术解决这些。

斑马鱼， 斑马鱼 ，已成为发育生物学的研究中广泛使用的生物。斑马鱼是属于鲤科鱼类热带，淡水脊椎动物。最初用于斑马鱼进行环境研究，以便对潜在的水污染物危害的信息，因为他们很容易获得，价格低廉，易于维护1。在过去的三十年里，斑马鱼已成为公认的主机的原因基因听话脊椎动物模型系统。斑马鱼表现与高等脊椎动物包括哺乳动物2,3高水平的解剖和生理保护。最近的基因组序列的注释显示，人类基因的大约70％的具有至少一个对应的斑马鱼4。例如，许多同源基因中发挥肾发育过程中的重要作用已经鉴定这些物种5-7之间。这DRAMATIC程度保护的问候细胞和分子生物学使研究人员能够在斑马鱼8创建模型，适用范围广的人类疾病，和斑马鱼已成为一个有价值的工具使用化学遗传筛选9，以确定潜在的药物治疗。 此外，斑马鱼模型不仅能够复述各种复杂的发育过程，而且是出现在人类疾病状态，但有几个附加的属性也提高它的吸引力作为模式生物的胚胎学研究。斑马鱼是卵生繁殖和利用广播产卵，这为研究者提供了从受精10的发作容易获得的胚胎。其它优点包括胚胎大小，透明度，以及快速，外部发展10。此外，斑马鱼的成年人具有高繁殖力和通常会产生比较大的离合器的尺寸是200-500之间的范围每个星期，最终使研究人员能够进行高通量的实验，如基于表型化工屏幕，轻松9。 即使如此，尽管优点由该斑马鱼模型表现出过多，有一些涉及到的分析和从早期发育阶段中得到的实验结果的沟通挑战。在一些情况下，斑马鱼胚胎的固有解剖可能模糊1的数据的可视化。受精后，最初的细胞经历多个切割事件随着开发的进展11。以下原肠胚形成，胚胎轴出现在tailbud阶段（10 HPF），使得它绕在蛋黄11。随后的发展过程通过分割期间，中继线将生长在质量和也由轴向伸长延长，使得沿着与蛋黄的一部分的尾囊本身从中央蛋黄质量延伸出11，在tailbud和20体节期（19 HPF）之间，尝试各种染色方法，如希望利用后，观察具体的发展特点，可以挑战既可视化和照片由于胚胎和不透明的几何形状卵黄囊。消除这些问题的方法之一是除去卵黄，然后压平胚胎成2维采样，可以在一个更简单的方式来分析。 下面的协议和视频资源提供如何成功地deyolk和平板装载胚胎以下固定和染色，使用一种或两种颜色WISH染色的例子的指南。愿望是一种广泛使用的技术，使之在保存标本特异性核酸的检测，从而允许对基因表达的被组织中检测到的网站（）。 WISH技术已被广泛描述，并且可以与各种颜色的基板以及进行感orescent检测系统12-20。在这里，我们提供我们用于发展的tailbud和分割阶段之间的斑马鱼胚胎修改WISH协议。另类WISH的协议，但是，与这里所描述的平坦安装程序兼容，如下面的协议一开始就指出。另外，平的安装可以利用在与任何数量的现有的可视化技术，如整装免疫组织化学，或细胞谱系跟踪标签，其未在此描述的协议一起使用。总体而言，平面安装的技术可以更好地使从实验的斑马鱼胚胎发育的早期阶段获得的数据的卓越分析和介绍。

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	<tbody>
		<tr height="68">
			<td height="68" style="height:68px;width:163px;">50 X E3</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">250 mM NaCl, 8.5 mM KCL, 16.5 mM CaCl2, 16.5 mM MgSO4 in distilled water. Supplement with methylene blue (1 x 10-5 M) (Sigma M9140) to inhibit contamination.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">1 X E3</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">Dilute 50 X E3 in distilled water.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">mesh tea strainer</td>
			<td style="width:96px;">English Tea Store</td>
			<td style="width:119px;">SKU #ASTR_KEN, MPN#1705</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">embryo incubation dish</td>
			<td style="width:96px;">Falcon</td>
			<td style="width:119px;">35-1005</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">transfer pipet</td>
			<td style="width:96px;">Samco</td>
			<td style="width:119px;">202, 204</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">flat-bottom microcentrifuge tube</td>
			<td style="width:96px;">VWR</td>
			<td style="width:119px;">87003-300; 87003-298</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">glass vial</td>
			<td style="width:96px;">Wheaton</td>
			<td style="width:119px;">225012</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">1 X Pbst</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">0.1% Tween-20 detergent in 1 X Pbs, made by diluting 10 X Pbs in distilled water.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Tween-20 stock</td>
			<td style="width:96px;">American Bioanalytical</td>
			<td style="width:119px;">AB02038</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">10X Pbs&#160;</td>
			<td style="width:96px;">American Bioanalytical</td>
			<td style="width:119px;">AB11072</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">paraformaldehyde</td>
			<td style="width:96px;">Electron Microscopy Services</td>
			<td style="width:119px;">19210</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:163px;">4% PFA/1X Pbs</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">Dissolve 4% PFA (w/v) in 1 X Pbs, bring to boil on a hot plate in a fume hood. Cool and freeze aliquots for storage at -20 &#176;C. Thaw just before use and do not refreeze stocks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">filter sterilization unit</td>
			<td style="width:96px;">Corning</td>
			<td style="width:119px;">430516</td>
			<td style="width:383px;">1L filter system, 0.45 um CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">MeOH</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">34860-4L</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">proteinase K</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">03-115-879-001</td>
			<td style="width:383px;">Dissolve in distilled water to make proteinase K stock (10 mg/mL) and store aliquots at -20 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">HYB+</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">50% formamide, 5X SSC, 0.1% Tween-20, 5 mg/mL yeast torula RNA, 50 ug/ul heparin</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:163px;">DIG/FLU labeled riboprobe in vitro transcription reaction reagents</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">11175025910; 11685619910</td>
			<td style="width:383px;">Refer to manufacturer instructions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">SP6 RNA polymerase</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">11487671001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">T7 RNA polymerase</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">10881775001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">T3 RNA polymerase</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">11031171001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">RNase 1 inhibitor</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">3335399001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">DNase 1 inhibitor, 10 X DNase 1buffer</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">4716728001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">glycogen</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">10901393001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">Ethanol (EtOH)</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">E7023</td>
			<td style="width:383px;">Aliquot 50 ml aliquots of 100% EtOH and 70% EtOH (diluted with molecular grade water) and store at -20 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">molecular grade distilled water</td>
			<td style="width:96px;">Mediatech</td>
			<td style="width:119px;">25-055-CM</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">waterbath</td>
			<td style="width:96px;">Thermo</td>
			<td style="width:119px;">51221073</td>
			<td style="width:383px;">Model 2831</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">nanodrop</td>
			<td style="width:96px;">Thermo</td>
			<td style="width:119px;">ND-2000c</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;"></td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">formamide</td>
			<td style="width:96px;">American Bioanalytical</td>
			<td style="width:119px;">AB00600</td>
			<td style="width:383px;">Store at -20 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">20X SSC</td>
			<td style="width:96px;">American Bioanalytical</td>
			<td style="width:119px;">AB13156</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:163px;">MAB</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">2L formula: Into 1.5 L of distilled water, mix 23.2 g maleic acid, 17.5 g NaCl, 55.0 g Trisma base, then add 8 mLs of 1M Tris-HCl pH 9.5, and then fill to 2 L. Autoclave.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">MABT</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">MAB + 0.1% Tween-20&#160;</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:163px;">block solution</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">We make in 50 ml fresh aliquots: 10 mLs of BSA (from 10% lab stock), 5 mls of FCS (from stock), and 35 mLs of MABT. Save unused block at 4 &#176;C to use for antibody solution. Note: Thaw the BSA and FCS stocks at 37 &#176;C&#8212;if you thaw at a higher temperature they become a thick gel (do not use).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">FCS (fetal calf serum) stock</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">16140089</td>
			<td style="width:383px;">Aliquot in 5 or 10 ml portions and store at -20 &#176;C.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:163px;">BSA (bovine serum albumin) stock</td>
			<td style="width:96px;">American Bioanalytical</td>
			<td style="width:119px;">AB00448</td>
			<td style="width:383px;">Make a 10% stock diluted in MAB by dissolving the BSA flakes at room temperature with rapid stirring, then store 10 ml aliquots at -20 &#176;C. Store undissolved BSA flakes at 4 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">anti-digoxigenin antibody</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">11-093-274-910</td>
			<td style="width:383px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">anti-fluorescein antibody</td>
			<td style="width:96px;">Roche</td>
			<td style="width:119px;">11-426-338-910</td>
			<td style="width:383px;">Store at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">12-well staining dish</td>
			<td style="width:96px;">BD-Falcon</td>
			<td style="width:119px;">35-3225</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">pre-staining&#160; buffer</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20. Always make fresh: it will precipitate in the course of a few days.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">staining solution-purple</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">For every 10mLs needed: add 45 uL of NBT and 35 uL of BCIP to fresh pre-staining buffer (not used on embryo samples).</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">staining solution-red</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">For every 10mLs needed: add 31.5 uL of INT and 35 uL of BCIP to fresh pre-staining buffer (not used on embryo samples)..</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">NBT stock</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">N6876</td>
			<td style="width:383px;">Stored at -20 &#176;C; powder diluted 50mg/mL in 70% DMF, 30% water.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">INT stock</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">I8377</td>
			<td style="width:383px;">Stored at -20 &#176;C; powder diluted 55mg/mL in 70% DMF, 35% water.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">BCIP stock</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">B8503</td>
			<td style="width:383px;">Stored at -20 &#176;C; powder diluted 50mg/mL in 100% DMF.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">DMF (dimethylformaldehyde)</td>
			<td style="width:96px;">American Bioanalytical</td>
			<td style="width:119px;">AB00450</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">glycine</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">G8898</td>
			<td style="width:383px;">&#160;0.1 M glycine, pH 2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">small plastic Petri dish</td>
			<td style="width:96px;">Corning</td>
			<td style="width:119px;">430589, 430588</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">glycerol</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">G7893</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">fine forceps</td>
			<td style="width:96px;">Roboz</td>
			<td style="width:119px;">RS-1050</td>
			<td style="width:383px;">Dumont Tweezers Pattern #55&#160;</td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:163px;">lash tool</td>
			<td style="width:96px;"></td>
			<td style="width:119px;"></td>
			<td style="width:383px;">Constructed by affixing a suitable lash to a pipet tip (sizes ranging from P10-P1000 can be used) using superglue. We use a naturally shed human eyelash, a naturally shed animal whisker or wiry fur coat hair, or a natural or synthetic lash purchased from the beauty department at a pharmaceutical or retail store (extra long lashes are most amenable to stable mounting on the pipet tip). The pipet tip is affixed onto a straight rod (8-10 cm in length) such as a needle holder (e.g. Fisher 08-965-A) for easy handling. See Figure 3 for images of these homemade tools.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">glass slide</td>
			<td style="width:96px;">Thermo-Fisher</td>
			<td style="width:119px;">4445</td>
			<td style="width:383px;">White Frost&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">glass coverslip</td>
			<td style="width:96px;">Thermo-Fisher</td>
			<td style="width:119px;">12-540A</td>
			<td style="width:383px;">18 x 18 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">modeling clay</td>
			<td style="width:96px;">Hasbro</td>
			<td style="width:119px;">Playdoh</td>
			<td style="width:383px;">Other craft modeling clay products can be substituted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">slide holder</td>
			<td style="width:96px;">Thermo-Fisher</td>
			<td style="width:119px;">12-587-10</td>
			<td style="width:383px;">Cardboard tray to store slides flat</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">stereomicroscope</td>
			<td style="width:96px;">Nikon</td>
			<td style="width:119px;">SMZ645, SMZ1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">compound microscope</td>
			<td style="width:96px;">Nikon</td>
			<td style="width:119px;">80i, 90i</td>
			<td></td>
		</tr>
	</tbody>
</table>

flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization

斑马鱼的胚胎是现在常用的基本和生物医学研究探讨发育过程的遗传控制和模拟先天性畸形。在生命的第一天，斑马鱼胚胎过程通过许多发展阶段，包括受精，卵裂，原肠胚形成，细分和结构，如肾脏，心脏和中枢神经系统的器官。一个年轻的斑马鱼胚胎的解剖结构呈现为参与许多活动的组织的可视化和分析一些挑战，因为胚胎在协会的发展具有圆形的蛋黄质量。因此，对于精确分析和实验的表型在tailbud和20体节阶段（10和19小时后受精（HPF），分别），例如那些使用染色之间固定的胚胎标本成像整体原位杂交（WISH），它通常希望从蛋黄B拆下胚胎所有的以及将它平放在载玻片上。但是，执行一台安装过程可能很乏味。因此，成功的和高效率的扁平安装准备通过剥离技术的视觉演示极大的便利，也有助于通过使用试剂，帮助在最佳的组织处理。在这里，我们为我们的愿望协议，用于一个或两个色检测在斑马鱼胚胎的基因表达，并演示如何在平坦的安装程序可以在一个固定染色标本的这个例子来进行。这种扁平的安装协议是广泛适用于在斑马鱼早期发育所出现的许多胚胎结构的研究，可以与固定胚胎样品进行染色等方法结合来​​实现。

在平坦的安装协议程序涉及从它的正常解剖形态，其中，所述本体轴线绕一个位于中心的卵黄球，放入一个二维制备（ 图1A）的斑马鱼胚胎的转化。年轻的斑马鱼胚胎整装成像是由胚胎轴是怎么缠蛋黄性质的限制。其结果是，整个侧面或背意见挂载染色胚胎标本只能捕捉到轴或晦涩的特定组织（ 图1B）的一部分。通过比较，deyolking胚胎和平坦化使得整个胚胎轴在同一时间（ 图1B）来实现可视化。这些限制是展示通过检查一个WISH染色的胚胎被标记的反义核糖核酸探针来检测irx3b（紫），这标志着相邻体节7至13中的肾祖细胞的一个子集，并MYOD1（红色），该标签的体节（ 图1B）。的irx3b +肾祖细胞的字段被遮住，在整个安装侧视图因为irx3b转录物大量存在于中枢神经系统，使肾字段实际上是不可能的可视化与横向照相机角度;进一步，当胚胎转动到背相机视图，因为该字段是缠的蛋黄（ 图1B）irx3b +肾祖细胞的字段是仅部分可见。然而，相同的胚胎下列平坦安装一个背视图使irx3b +肾祖细胞中相对于沿躯干和其它胚胎结构（ 图1B）的体节一个直接的方式被分析的整个领域。因此，精心的空间域可以理想地证明用这种方法。 参与的成功执行的平板安装过程的几个主要步骤。要执行此techniquE，卵黄球必须首先从胚胎中适当的（ 图2A），它可以与精密仪器，如一双细镊子来完成，而胚胎是使用立体显微镜可视化分离。以下将粗除去卵黄球，细睫毛工具被用来刮掉已经保持附着在胚胎（ 图2B）卵黄颗粒。的剩余卵黄颗粒的去除有助于促进胚胎组织的可视化。最后，deyolked胚胎被操纵以在载玻片上（ 图2C），这使得使用图像处理软件对样品的观察和辅助文件和附连到显微镜的照相机稳定地定位。睫毛工具可以安装到手柄上，如果需要（ 图3A，材料表），可以通过使用强力胶固定用一个合适的睫毛在移 ​​液 ​​管尖被构造自制设备，。不同的睫毛样品可以采购到生产睫毛的长度和锥度（ 图3B），每个用户可能有不同的偏好，一旦他们开始尝试与平板安装程序上略有不同特性的工具。睫毛样品的实例包括自然脱落人类的睫毛，自然脱落的动物弦毛或晶须，​​最后合成品种市售睫毛的化妆品零售部门发现。 周围和含有定位在载玻片上（ 图4A）的样品（次）局部区域进行成像的高分辨率分析。探针的结合被用来标记在显影肾脏祖细胞是引起肾脏在野生型胚胎在具有双色WISH早期发育阶段，然后再扁平装入已完成制剂可以看到的试样（ 图4B，4C ）。在早期的体节阶段，肾祖细胞是由第划定在一个U形图案pax2a成绩单（紫色）围绕近轴中胚层的伴随表示三角形C（DLC）（红色）（左边的列， 图4B）6,7的EIR表达。相比较而言，当检测到与紫色基板DLC转录物和被标记的与后脑标记猪Krox20（红色）的组合，使肾祖细胞的喙子域是可视化表达DLC（右列中， 图4B），如前面所指出的6,7。平板安装制剂可用于类似的研究后体节形成阶段。在14体节阶段，我们分析了肾脏标志物pax2a，SLC4A4，SLC12A3和（紫色）随着smyhc1（红色），这标志着体节（ 图4C）的表达。这些组合能够与pax2a整个肾脏祖域的映射关系，同时揭示了细胞的一个子域喙expr的一个子集essed slc4a4a和由该表达SLC12A3肾脏祖细胞的一个非重叠的尾部子域进行区分。 双色WISH和平坦装载制剂也可用于蜂窝结构域的器官在斑马鱼与来自环境暴露于小分子6,7（ 图5）的基因突变或其他扰动在研究有价值。斑马鱼和NLS突变体库的醛脱氢酶1A2（ALDH1A2，原名RALDH2），它编码所需的维甲酸（RA）的生物合成的酶怀有突变。 RA是许多肾细胞类型，包括足细胞，有助于使胚胎肾的血液过滤器，被称为肾小球6,7形成的正常发育是必不可少的。愿望是执行以伴随与T的分界标记足细胞祖细胞与wt1a他显影体节与smyhc1和后脑与在野生型胚胎中猪Krox20，NLS突变体胚胎中， 解放突变体胚胎中，并用ALDH酶的化学抑制剂，DEAB（ 图5）处理的胚胎。胚胎缺乏ALDH1A2表达量减少wt1a表达与野生型的胚胎，而DEAB处理的胚胎表现出wt1a成绩单的废止（ 图5，右列 ）。两者合计，这些有代表性的结果展示如何在平面安装技术可以用于分析和记录的精度在早期胚胎解剖上的差异，因此对于有价值的发育研究来实现。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51604/51604fig1highres.jpg" src="/files/ftp_upload/51604/51604fig1.jpg" /> 平板安装准备相图1。概述配给斑马鱼胚胎。 A）平面安装的过程使沿胚轴组织同时认为，因为中央的蛋黄质量被删除，胚胎位置稳定在一个平面上。一个整装乙）图片如欲染胚胎在外侧和足背的意见，再经过一台安装准备进行。胚胎被染了反义RNA探针来检测编码irx3b（紫色）和MYOD1（红色）基因转录。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51604/51604fig2highres.jpg" src="/files/ftp_upload/51604/51604fig2.jpg" /> 图2原理平面安装程序斑马鱼胚胎。 A）将胚胎第一极deyolked以除去大部分的蛋黄质量，则（B）的腹侧表面被精细deyolked以除去剩余的卵黄颗粒，并洗完后，胚胎是（C）安装背侧上进行直观分析和/或感光成像的载玻片。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51604/51604fig3highres.jpg" src="/files/ftp_upload/51604/51604fig3.jpg" /> 图3的例子睫毛工具照片相比，细镊子。 A）自制睫毛工具进行成像旁边一个标准的一双细镊子，位于靠近米尺提供一定的参考。 二）睫毛工具旁边的细镊子放大视图，沿视图的顶部提供了参考毫米。两种不同的睫毛工具同时显示，以用来标记从获得的睫毛星号（*）一个自然脱落的人睫毛，和两个星号（**）用于标记从自然脱落的猫晶须获得并修剪成一个睫毛做一个有点钝端。在每个CA本身，间隙是通过移液管尖的螺纹，并与瞬间粘合剂的几个外套固定到逐渐形成强大的密封件，以稳定/锚睫毛至附连到处理设备的吸液管。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51604/51604fig4highres.jpg" src="/files/ftp_upload/51604/51604fig4.jpg" /> 图4。表征的在野生型斑马鱼胚胎中的肾脏祖字段的发展变化。 A）滑动示意图指示（B，C）B）野生型胚胎在1，3，5体节期是由双色愿望染色来评估肾祖场，给出的组成成像分析领域上升到胚胎肾，或鱼头。 （左栏）编码转录因子pax2a（染紫色）成绩单标记几个人群在胚胎，包括肾progenitor字段出现从中间胚层。成绩单编码Notch配体DLC标记，从近轴中胚层（染为红色）形成的体节。 （右列）当DLC稿（染色紫色）的表达和后脑菱标记猪Krox20（染成红色）进行检查以同样的胚胎，DLC表达可以在相邻体节的肾脏祖细胞的喙子域观察1-5，这是在共染色DLC与pax2a C）的野生型胚胎在14体节期模糊是双重或三重染色与肾标记（紫色的组合），体节标记smyhc1（红色）和后脑菱标记猪Krox20（也红了）。 （上图）在这个阶段，pax2a成绩单继续划定肾脏祖细胞。 （中，下板）在肾祖的领土，一个喙子域被标记由SLC4A4和SLC12A3编码的转录标记尾鳍子域。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51604/51604fig5highres.jpg" src="/files/ftp_upload/51604/51604fig5.jpg" /> 图5：采用平板安装准备工作，以表征引起基因突变或调节视黄酸生物合成的化学基因扰动的表型。在wt1a的15体节期的意愿表达模式（紫色）和smyhc1/krox20（红色）进行比较野生型和胚胎与维甲酸（RA）的生产缺陷之间由于醛脱氢酶1A2（NLS和lib突变）或视黄醛脱氢酶活性与二乙氨基苯甲醛（DEAB）化学抑制缺陷。 RA的生产库的减少是稍微比NLS更严重，例如该库的胚胎表达wt1a成绩单略有减少染色比同样上演NLS突变的胚胎，而DEAB治疗野生型与wt1a表达完整废除有关。

Watch this Scientific Journal Video about Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization at JoVE.com

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

斑马鱼的胚胎是发育生物学研究的一个很好的模型。胚胎发育过程中，斑马鱼发展与蛋黄质量，其中介绍的样品观察和分析，三维的挑战。本协议描述了如何创建原位 （WISH）染色斑马鱼胚胎标本整装二维平板安装的准备工作。

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

flat-mount-preparation-for-observation-analysis-zebrafish-embryo

Research

JoVE Journal

Biology

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

24.1K 次观看.  University of Notre Dame. 斑马鱼的胚胎是发育生物学研究的一个很好的模型。胚胎发育过程中，斑马鱼发展与蛋黄质量，其中介绍的样品观察和分析，三维的挑战。本协议描述了如何创建原位 （WISH）染色斑马鱼胚胎标本整装二维平板安装的准备工作。 

视频: Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount 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

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 ...

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

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

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

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

使用全山原位杂交分子和有机体生物学链接

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

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

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

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

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

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

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

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

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

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

全山原位杂交E8.5，E11.5小鼠胚胎

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

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 ...

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 ...