1。整个安装原位杂交斑马鱼胚胎 1.1胚胎的制备<ol><li>收集胚胎发育阶段所需。请参阅基梅尔等 5萌芽阶段的描述。 </li><li>删除手动绒毛膜使用钳（杜蒙列表镊子5号）。 </li><li>修复胚胎在4％的多聚甲醛（PFA）的PBS中的溶液（磷酸盐缓冲盐水），在4℃下过夜</li><li>胚胎用PBS洗净，每次5分钟，在室温下至少3倍。 </li><li>商店胚胎在-20°C在100％甲醇（甲醇），直到用于未来应用原位杂交技术，包括整个安装。 </li><li>冻结上演胚胎总RNA的提取和存储在液氮中，在-80℃下</li><li>上演胚胎中提取RNA合成cDNA进行PCR扩增和随后的克隆。 </li></ol&gt; 1.2合成地高辛标记的RNA探针<ol><li>探针模板的PCR扩增和克隆。设置，总体积为50μl的PCR反应，如表1中所示。使用基因组DNA或更小的cDNA为模板，在PCR反应中。包括在72℃10分钟的延伸时间的最后一个周期，以确保PCR反应产物的全长和3&#39;端腺苷化，以促进TOPO克隆。 </li></ol><table border="1"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td>模板DNA </td><td> 100毫微克</td></tr><tr><td> PCR缓冲液（10X） </td><td> 5微升</td></tr><tr><td> dNTPs浓度（10毫米） </td><td> 2微升</td></tr><tr><td>引物（10毫米） </td><td>各1μM </td></tr><tr><td>加水至最终体积为</td><td> 49.7微升</td></tr><tr><td> Taq聚合酶（5单位/＆＃956升） </td><td> 0.3微升</td></tr><tr><td> 反应体积 </td><td> 将50μl </td></tr></tbody></table> 表1中。PCR扩增反应。 <ol start="2"><li>验证的大小的PCR产物用琼脂糖凝胶电泳。的估计大小的PCR产物为探针的合成是接近1 kb的。 </li><li>特别要注意避免核酸开展实验，无菌条件下使用无核酸水的污染。在观察多个产品，凝胶净化适当大小的PCR产物，然后再继续使用TOPO TA克隆试剂盒克隆。优化PCR反应，以消除拖尾的外观和多个频段。 </li><li>下进行，如表2中所示的TOPO克隆反应。联合TOPO载体和PCR产品，并在室温下孵育5分钟。 </li></ol><t能够边境="1"&gt; <tbody><tr><td> 试剂 </td><td> 化学感受细胞 </td><td> 电感受态细胞 </td></tr><tr><td>新鲜的PCR反应产品</td><td> 1-4微升</td><td> 1-4微升</td></tr><tr><td>盐水</td><td> 1微升</td><td></td></tr><tr><td>稀释盐溶液</td><td></td><td> 1微升</td></tr><tr><td>水</td><td>总成交量5微升</td><td>总成交量5微升</td></tr><tr><td> TOPO载体</td><td> 1微升</td><td> 1微升</td></tr><tr><td> 最终成交量 </td><td> 6微升 </td><td> 6微升 </td></tr></tbody></table> 表2中。TOPO克隆反应。 <ol start="5"><li> TOPO克隆产品纳入/引入到一杆能干细胞。 Ť他的过程被称为转型。 </li><li>转化的细胞在37℃下孵育至少1小时，以260 rpm以确保表达的抗生素抗性基因。板预热的Luria Bertani（LB）琼脂平板，含50μg/ ml氨苄青霉素和X-gal的转化混合物（50-100微升）。孵育37℃过夜。 </li><li>确定插入基于外观白色或淡蓝色菌落的存在。 TOPO载体包含lacZ基因编码β-半乳糖苷酶。在X-gal的存在下，β-半乳糖苷酶的生产形成蓝色。产生的DNA片段连接到质粒破坏了形成功能性β-半乳糖苷酶和白色菌落。白色菌落或浅蓝色殖民地的确认存在的插入。 </li><li>挑选白色菌落琼脂平板上，用无菌枪头，将它们放置在无菌的10毫升管含4毫升的LB培养基和培养，在37°C，在晃动的温浴器以200rpm过夜。 </li><li>纯化的质粒DNA，用质粒DNA的试剂盒，根据制造商的指示。 </li><li>线性化的cDNA以下质粒纯化每个探头，有一个独特的站点位于3&#39;（感探头）或5&#39;（反义探针）插入适当的限制性内切酶消化5微克的cDNA。 </li><li>纯化的线性化的DNA用酚/氯仿抽提方法。加入等体积的苯酚/氯仿，顶点大力，持续30秒，旋转3分钟，在4℃下以13,000 rpm上层水相转移到干净的试管中，并加入等体积的氯仿。在4℃下以13,000 rpm的3分钟的自旋上层水相转移到干净的试管。加入10％的醋酸钠（3米）和2.5倍体积的100％乙醇沉淀DNA，并储存于-20°C过夜。在4℃下以13,000 rpm 15分钟自旋除去乙醇，并用75％乙醇洗涤沉淀。该协议可以停止和DNA可以被ST在-20°C相或第二天恢复。在4℃下以10,000 rpm 10分钟自旋小心除去上清液，持续2分钟，在室温下干燥沉淀。将沉淀重悬于10μl的DEPC处理过的水和孵育在55℃下20分钟。量化DNA浓度用紫外分光光度计。 </li><li>通过琼脂糖凝胶电泳，使用1μl的DNA在1％琼脂糖凝胶上运行约30分钟，评估纯度。 </li><li>设置RNA贴标反应。用无RNA酶的反应瓶中，加入1-5微克纯化的DNA模板，无菌的，无RNase的DEPC处理过的双蒸水至体积为13微升。冷藏冰和反应小瓶中添加下列试剂（ 见表 3）: </li></ol><table border="1"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> NTP标记混合物（10倍） </td><td> 2微升</td></tr><tr><td>转录的buffER（10倍） </td><td> 2微升</td></tr><tr><td>保护核糖核酸酶抑制剂</td><td> 1微升</td></tr><tr><td> RNA聚合酶SP6 或 </td><td></td></tr><tr><td> RNA聚合酶T7 </td><td> 2微升</td></tr><tr><td> 最终成交量 </td><td> 20μl的 </td></tr></tbody></table> 表3中。，RNA标记反应。 <ol start="14"><li>轻轻地混合试剂，然后以2,000 rpm离心收集在底部，持续30秒，在室温下孵育2小时，将反应混合物在37℃。 </li><li>加入2微升的无RNA酶的DNA酶I。下孵育30分钟，在37℃，并通过加入2微升的0.2M EDTA（pH 8.0）中，使反应停止，以去除模板DNA。 </li><li>通过加入2.5微升4M的LiCl和75微升100％的乙醇，沉淀出反应产物。混合解决方案，并保持在-20℃，45分钟Øŗ存放于-20℃过夜和协议恢复第二天。 </li><li>在4℃下以13,000 rpm离心悬浮液30分钟，然后用500μl的70％乙醇再延长10分钟，2分钟干燥储存在-20℃，离心，洗涤沉淀，重悬在50μl孵育在37℃下在30分钟</li><li>验证探针的质量和完整性，约30分钟，在1％琼脂糖凝胶上运行1-2毫升。这也证实了在预期的大小在凝胶上运行的单品的存在下。 </li><li>量化的探针浓度，使用分光光度计测定RNA的量。从该反应中的产率是约10μg的RNA（100〜200毫微克/微升）。 </li></ol> 1.3全安装在原位杂交<ol><li> 第1天 <ol><li> 补液 <ol><li>准备使用尼龙筛（孔径大小小于1毫米）和Eppendorf管的篮子。构建筐切割机克Eppendorf管中（1.5毫升）以除去的锥形端部。要找出个别篮子使用不同颜色的管子，并卸下前轮圈其中一半。 </li><li>切小块尼龙网覆盖的Eppendorf管的切割端。通过在通风橱中的电热板之间设置到中高加热棒的管的切割端的尼龙网眼。冷却后，除去过量的尼龙网丝从筐，并将其储存在室温下在100％甲醇中。 </li><li>使用第3由100％的六孔板的孔中，然后，准备连续稀释在PBS（75％[体积/体积的甲醇，50％[体积/体积]甲醇和25％[体积/体积]甲醇）的甲醇PBST（磷酸盐缓冲液含0.1％吐温20），在接下来的3个井。 </li><li>待胚胎在原位杂交程序使用定制的篮图A 1a和1b所示。使用RNase-free的解决方案的杂交重刑在6孔培养板中的步骤（4毫升/孔），为了实现这一目标，将第一阱中的6筐。地点篮与RIM首先由轮辋连续排列的不同颜色的篮子没有。 6的基因表达模式使用这种安排，可同时测定。 </li><li>将脱水相同的胚胎发育阶段到相同的购物篮（ 参见 图1）。 </li><li>的移动筐在6孔板以及从一个到下一个的每个步骤（5分钟）再水合胚胎。使用六口井填充适当的缓冲。胚胎每个稀释度进行一次。在这种方式中，胚胎洗涤6次，5分钟/洗涤。补液脱水胚胎是通过用PBS代替甲醇，然后由100％PBST洗涤3倍。 </li></ol></li><li> 通透性月刊用蛋白酶K（10微克/毫升），在室温下再水化的胚胎，使它们可渗透的，如表4中所示</st荣&gt;。 <table border="1"><tbody><tr><td> 萌芽阶段（小时受精后） </td><td> 消化期（蛋白酶K） </td></tr><tr><td>最多6个</td><td> 15秒</td></tr><tr><td> 6-12 </td><td> 30秒</td></tr><tr><td> 12-18 </td><td> 3分钟</td></tr><tr><td> 24 </td><td> 12-15分钟</td></tr><tr><td> 48 </td><td> 25-30分钟</td></tr><tr><td> 72 </td><td> 40分钟</td></tr><tr><td> 96-120 </td><td> 50分钟</td></tr></tbody></table> 表4中。通透反应。 </li><li> Postfixation和PBST清洗 <ol><li>修复的胚胎，在4％多聚甲醛（重量/体积），在1x PBS中孵育20分钟。 </li><li>去除残留的多聚甲醛洗涤胚胎3X，5分钟/洗，在1x PBST。 </li></ol> &lt;/ LI&gt; <li>准备的乙酰乙酰化混合物（125微升三乙醇胺和27微升的醋酸酐10毫升DDH 2 O）和孵化胚胎两次，每次10分钟。为了最大限度地减少内源性磷酸酶活性，准备乙酰混合物新鲜时需要。要去除多余的试剂，两次胚胎在PBST洗（10分钟/洗涤） </li><li> 的预杂交 Prehybridize胚胎筐胚胎转移到1.5毫升无菌的Eppendorf管中，用200μl的杂交混合物孵育2-4小时，在70℃的水浴中。 </li><li> 预吸附的抗体从篮子中取出约50个胚胎，并将它们传送到1.5毫升无菌Eppendorf管。用抗DIG-AP抗体（1:500）用封闭缓冲液（PBST中1个，2％小牛血清[体积/体积]，2毫克/毫升牛血清白蛋白的相互作用[BSA]），在室温下2-4小时，孵育然后将其存储在4℃过夜。从孵化器中删除在早上ð允许胚胎达到室温。 </li><li> 杂交添加100-200纳克的探针到预杂交胚胎和孵育过夜，在70℃水浴中。 </li></ol></li><li> 第2天 <ol><li> 盐水柠檬酸钠（SSC）洗 <ol><li>取50毫升的试管杂交的混合物（HM），:2X SSC和0.2倍SSC在烧杯中的水在70℃水浴中，他们至少20分钟prewarm。删除含有该探针的杂交反应混合物中，加入1 ml预热的杂交混合物的管，并孵育15分钟，在70℃下填充六孔板预热的HM的2×SSC中与各种稀释度的替换100％通过50％至25％HM HM，以除去过量的试剂。 </li><li>在等待的同时，填补六孔板2倍SSC洗涤，并保持在70°C。 </li><li> 15分钟后，这里的胚胎从管保持在6孔板中填充稀释篮子HM s的具有2×SSC洗杂交胚胎购物篮以及从一个移动到下一个（15分钟，在70℃水浴中）。洗涤胚胎2倍，15分钟/洗涤，在100％的2×SSC在70℃下</li><li>置另一水浴中在65℃下填补六孔板高严谨的0.2×SSC中洗涤，以避免非特异性的转录与探针杂交。 </li><li>胚胎后洗涤两次用2×SSC在70℃下，按照进一步在0.2×SSC中洗涤30分钟，在65℃下</li></ol></li><li> 用PBST洗涤准备六孔板与连续稀释的0.2×SSC在PBST如下:75％（体积/体积）0.2×SSC，50％（体积/体积），0.2×SSC，25％（体积/体积）的0.2倍SSC，和剩余的两个阱100％PBST。清洗篮移动以及从一个到下一个（5分钟，在室温下为每个步骤使用摇杆）杂交的胚胎。 </li><li> 预孵育垫上预温育胚胎在室温下3-4小时BLO他妈的缓冲区（1X PBST，2％小牛血清体积/体积]，2毫克/毫升BSA）。 </li><li> 抗地高辛抗体孵育胚胎用抗DIG-AP抗体（1:5000）的溶液中，在封闭缓冲液，在4℃下轻轻摇动过夜孵育 。 </li></ol></li><li> 第3天 <ol><li> PBST清洗准备六孔板，4毫升/ PBST。孵化的胚胎洗净篮子移动在6孔板PBST 15分钟期间的6倍。胚胎可以储存在4℃下，继续第二天的协议。 </li><li> Prestaining Prestain 2个15分钟洗涤用缓冲液，在室温下轻轻摇动prestaining BCIP（5 -溴-4 -氯-3 -吲哚基磷酸酯）和NBT（氮蓝四唑）的存在下在孵化前的胚胎。 BCIP和NBT是用于碱性磷酸酶活性的发色底物。由于探针附着碱性磷酸酶日紫色表示ê发展目标基因的表达。胚胎可以储存在4℃下，继续第二天的协议。 </li><li> 染色法 <ol><li>在室温下，在黑暗中与BCIP及NBT 30分钟孵育胚胎。 </li><li>每30分钟使用立体显微镜监测染色反应。反应时间从15分钟，16小时，取决于基因表达水平变化。反应时间更短和更长的高表达基因的为弱表达基因。感探测器将探测信号，如果感兴趣的基因表达互补反义（AS）成绩单。 </li></ol></li><li> 停止反应后达到所需的染色强度，使反应停止，转移到孔中，反应终止液（1倍的PBS液，pH5.5，1mM EDTA的溶液，0.1％吐温）含有胚胎的筐。这将避免颜色的进一步发展。的胚胎冲洗10分钟，2倍摇杆温柔AG在室温下itation。 </li><li> Postfixation <ol><li>孵育20分钟的多聚甲醛的PBS中的4％（重量/体积）中的胚胎。 </li><li>用PBST洗的胚胎（5分钟/洗涤）三次，以除去残留的多聚甲醛。在4°C，存储固定的胚胎在黑暗的一站式解决方案</li></ol></li><li> 安装，观测和图像采集 <ol><li>胚胎转移到一个六孔板中，含有100％的甘油，使用可能的最小体积的PBS。将6孔板在摇臂上，轻轻地在黑暗中在室温下搅拌过夜。 </li><li>放置在100％甘油中的胚胎，然后在立体显微镜下观察信号。 </li><li>获取图像，并保存为Tiff文件格式或任何其他类型可以生产高品质的图像，例如Photoshop（ 图3-6），通过使用图像处理软件。 </li></ol></li></ol></li></ol>I N原位杂交<ol><li>来实现双标记的方法如上所述从1.1 - 1.3.1.7使用，但包括地高辛，荧光素标记的探针在杂交过程中同时孵育。 </li><li>按照同样的步骤，杂交后洗即SSC和的PBST洗涤（步骤1.3.2.1和1.3.2.2）和阻塞孵化（步骤1.3.2.3）。执行顺序如下文所述的抗体和染色反应。 </li><li>孵育胚胎AP耦合抗地高辛抗体（1:5000），在4℃，轻轻搅拌。 </li><li>洗净和孵化BCIP-NBT（的步骤1.3.3.1 - 1.3.3.6）。 </li><li>检测后，洗两次胚胎用PBST，在室温下20分钟。 </li><li>在PBST中，在65℃下孵育胚胎用1mM EDTA的溶液30分钟，以灭活的抗地高辛抗体。 </li><li>在100％甲醇孵育胚胎其次在75％，50％，25％甲醇机智补液ħPBST PBST 1小时。 </li><li>孵育过夜AP耦合荧光素（1:2,000）封闭液在4℃，轻轻摇动的胚胎。 </li><li>清洗胚胎用PBST（步骤1.3.3.1）和染色前的胚胎，2次15分钟与预染色缓冲液（0.1米的Tris-盐酸，pH为8.2）在室温下，轻轻摇动，在孵化前的存在下固红。协议可以被停止和胚胎可以被存储在4℃下轻轻摇动，恢复第二天。 </li><li>一个快速红色片剂在2毫升染色缓冲液使用前立即溶解。 </li><li>启动耐晒红试剂染色缓冲胚胎的染色反应。 </li><li>停止时达到所需的染色强度的染色反应。这可能需要从几个小时，在室温下染色时，在4℃，过夜一天或两天立即停止反应，如果染色强度不会进一步发展或开始出现非特异性性时相比，检测控制。 </li><li>按照上述程序的其余步骤即固定后，安装，观测和图像采集（steps1.3.3.5和1.3.3.6）。 </li></ol>

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8, 353-367 (2007).</li><li>Wolman, M., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+genetics+in+larval+zebrafish:+learning+from+the+young.">Behavioral genetics in larval zebrafish: learning from the young.</a> Dev. Neurobiol. 72, 366-372 (2010).</li><li>Lawson, N. D., Wolfe, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+for+the+analysis+of+vertebrate+development+in+the+zebrafish.">Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish.</a> Dev. Cell. 21, 48-64 (2011).</li><li>Arkhipova, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+regulation+of+the+hb9/mnx1+beta-cell+progenitor+specific+enhancer+in+zebrafish.">Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.</a> Dev. Biol. 365, 290-302 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Chitramuthu, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+and+embryonic+expression+of+zebrafish+PCSK5+co-orthologues:+functional+assessment+during+lateral+line+development.">Molecular cloning and embryonic expression of zebrafish PCSK5 co-orthologues: functional assessment during lateral line development.</a> Dev. Dyn. 239, 2933-2946 (2010).</li><li>Liao, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCL/Tal-1+transcription+factor+acts+downstream+of+cloche+to+specify+hematopoietic+and+vascular+progenitors+in+zebrafish.">SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish.</a> Genes Dev. 12, 621-626 (1998).</li><li>Stainier, D. Y., Weinstein, B. M., Detrich, H. W., Zon, L. I., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloche,+an+early+acting+zebrafish+gene,+is+required+by+both+the+endothelial+and+hematopoietic+lineages.">Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages.</a> Development. 121, 3141-3150 (1995).</li><li>Detrich, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraembryonic+hematopoietic+cell+migration+during+vertebrate+development.">Intraembryonic hematopoietic cell migration during vertebrate development.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 10713-10717 (1995).</li><li>Liao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+gene+cloche+acts+upstream+of+a+flk-1+homologue+to+regulate+endothelial+cell+differentiation.">The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.</a> Development. , 124-381 (1997).</li><li>Krauss, S., Concordet, J. P., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functionally+conserved+homolog+of+the+Drosophila+segment+polarity+gene+hh+is+expressed+in+tissues+with+polarizing+activity+in+zebrafish+embryos.">A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos.</a> Cell. 75, 1431-1444 (1993).</li><li>Akimenko, M. A., Ekker, M., Wegner, J., Lin, W., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+expression+of+three+zebrafish+genes+related+to+distal-less:+part+of+a+homeobox+gene+code+for+the+head.">Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head.</a> J. Neurosci. 14, 3475-3486 (1994).</li><li>Alexander, J., Rothenberg, M., Henry, G. L., 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=casanova+plays+an+early+and+essential+role+in+endoderm+formation+in+zebrafish.">casanova plays an early and essential role in endoderm formation in zebrafish.</a> Dev. Biol. 215, 343-357 (1999).</li><li>Milewski, W. M., Duguay, S. J., Chan, S. J., Steiner, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+PDX-1+structure,+function,+and+expression+in+zebrafish.">Conservation of PDX-1 structure, function, and expression in zebrafish.</a> Endocrinology. 139, 1440-1449 (1998).</li><li>Argenton, F., Walker, M. D., Colombo, L., Bortolussi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+the+trout+insulin+promoter:+implications+for+fish+as+a+favorable+model+of+pancreas+development.">Functional characterization of the trout insulin promoter: implications for fish as a favorable model of pancreas development.</a> FEBS Lett. 407, 191-196 (1997).</li><li>Biemar, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+development+in+zebrafish:+early+dispersed+appearance+of+endocrine+hormone+expressing+cells+and+their+convergence+to+form+the+definitive+islet.">Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet.</a> Dev. Biol. 230, 189-203 (2001).</li><li>Thisse, C., Thisse, B., Schilling, T. F., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+zebrafish+snail1+gene+and+its+expression+in+wild-type,+spadetail+and+no+tail+mutant+embryos.">Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.</a> Development. 119, 1203-1215 (1993).</li><li>Cadieux, B., Chitramuthu, B. P., Baranowski, D., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+progranulin+gene+family+and+antisense+transcripts.">The zebrafish progranulin gene family and antisense transcripts.</a> BMC Genomics. 6, 156 (2005).</li><li>Chitramuthu, B. P., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+zebrafish+and+knockdown+technology+to+define+proprotein+convertase+activity.">Use of zebrafish and knockdown technology to define proprotein convertase activity.</a> Methods Mol. Biol. 768, 273-296 (2011).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+in+situ+hybridization+on+whole-mount+zebrafish+embryo.">High resolution in situ hybridization on whole-mount zebrafish embryo.</a> Nat. Protoc. 3, 59-69 (2008).</li></ol>

作者宣称，他们有没有竞争经济利益。

我们已经开发了改进的方法具有高的分辨率的可视化RNA。的原位杂交程序，进行了使用简单的量身定制筐与多孔底的（图1）。胚胎的处理，在无菌条件下，在6孔培养板中，在室温下使用RNase-free的解决方案。 是由不同颜色的Eppendorf管中，以分隔胚胎筐。筐带或不带的轮辋是用于容易取向，并确定为对应于一个特定的探针的购物篮内的胚胎。 <p class="jove_c...

斑马鱼（ 斑马鱼 ）已经成为一个强大的动物模型，为研究脊椎动物的发育，疾病，行为，以及在药物筛选1-3。斑马鱼的胚胎，可以得到大量从一个单一的十字路口。测土配方施肥和发展发生子宫外和透明的胚胎发育迅速。关键发育事件发生在第一受精后48小时（HPF）。这包括器官原基的外观和细胞分化的启动。击倒或过度表达的蛋白质可以通过在吗啉代反义寡核苷酸（MO）胚胎的显微注射，或上面的一个或两个细胞阶段（0.75 HPF）的分别的mRNA。 斑马鱼胚胎的愿望，有利于利益的特定基因的时空表达的研究。后的愿望技术应用显微注射胚胎mRNA或MO过表达式ESS或击倒特定的蛋白质水平揭示微分调节其他基因。 可以是相关的基因表达模式的变化与表型的变化，在器官发生早期揭示的靶基因的功能。由于探头可以预先制备和贮存，原位杂交技术可以应用在一个时间使用六孔板和定制的篮子，以显示至少20个基因的基因表达模式。

<table border="0" cellpadding="0" cellspacing="0" width="1117">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:440px;">Taq Polymerase</td>
			<td style="width:159px;">Invitrogen</td>
			<td align="right" style="width:137px;">10342053</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TopoTA Cloning Kit- Dual Promoter pCRII-TOPO</td>
			<td>Invitrogen</td>
			<td>K4650-01</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HQ Mini Plasmid Purification Kit</td>
			<td>Invitrogen</td>
			<td>K2100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Roche</td>
			<td style="width:137px;">11 685 660 001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Not1 and HindIII Restriction Enzymes</td>
			<td>Invitrogen</td>
			<td>15441-025, 15207-012</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;">Buffer Saturated Phenol, ultrapure</td>
			<td>Invitrogen</td>
			<td align="right">15513039</td>
			<td>Store at 4 &#176;C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Chloroform</td>
			<td>Fisher</td>
			<td>C607-1</td>
			<td>Toxic and suspected carcinogen. Work under fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free DNAse I</td>
			<td>Roche</td>
			<td align="right">4716728001</td>
			<td>Prepare small aliquots and store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase</td>
			<td>Roche</td>
			<td align="right">11277073910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase Inhibitor</td>
			<td>Roche</td>
			<td>10777-019</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.0 M Sodium Acetate (pH 5.5)</td>
			<td>Fisher</td>
			<td>50-751-7355</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">100% Ethanol</td>
			<td>Commercial alcohols</td>
			<td style="width:137px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Diethyl Pyrocarbonate (DEPC)</td>
			<td>Sigma</td>
			<td>40718-25ML</td>
			<td style="width:383px;">DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Fisher</td>
			<td>SS255-1</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA 0.5 M (pH 8.0)</td>
			<td>Fisher</td>
			<td>50-751-7404</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean Salt</td>
			<td>Aquarium Systems</td>
			<td style="width:137px;">N/A</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td>Fisher</td>
			<td style="width:137px;">FL-03-0900</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148-1KG</td>
			<td>Prepare and store at -20&#176;C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Phenylthiocarbomide (PTC)</td>
			<td>Sigma</td>
			<td>P7629-25G</td>
			<td>PTC is highly toxic. Use safety glasses, gloves, and dust mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Fisher</td>
			<td>A947-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont (Watchmaker&#8217;s) Forceps pattern no. 5</td>
			<td>Fine Science Tools</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Six-well Cell Culture Cluster</td>
			<td>Sarstedt</td>
			<td align="right" style="width:137px;">83.1839</td>
			<td></td>
		</tr>
		<tr height="182">
			<td height="182" style="height:182px;">Baskets are made of nylon mesh and Eppendorf tubes</td>
			<td>In-house preparation</td>
			<td style="width:137px;"></td>
			<td style="width:383px;">To make the baskets, cut Eppendorf tubes (1.5 ml) with or without rims to remove the conical end. Cut nylon mesh into small pieces corresponding to the size of&#160;the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical&#160;hot plate until both the tube and nylon mesh stick together (carry out in fume hood). Cut off the excess mesh from the baskets and store them in 100% methanol until used.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Polyoxyethylenesorbitan Monolaurate (Tween 20)</td>
			<td>Sigma</td>
			<td>P1379-500ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic Anhydride</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">242845</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethanolamine</td>
			<td>Sigma</td>
			<td>90279-100ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide, high purity grade</td>
			<td>Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">AG501-X8 Resin</td>
			<td>Bio Rad</td>
			<td style="width:137px;">142-6424</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric Acid monohydrate</td>
			<td>Sigma</td>
			<td>C0706-500G</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin Sodium Salt</td>
			<td>Sigma</td>
			<td>H3393-25KU</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline-sodium Citrate Buffer (SSC)</td>
			<td>Sigma</td>
			<td style="width:137px;">S6639</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tRNA from bakers yeast type X</td>
			<td>Sigma</td>
			<td>R5636-1ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:440px;">NaCl</td>
			<td>Fisher</td>
			<td>S640-500</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher</td>
			<td>BP153-1</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td>Fisher</td>
			<td>S25403</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Levamisole Hydrochloride</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">31742</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:440px;">N,N-Dimethylformamide 
			anhydrous (DMF)</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">227056</td>
			<td style="width:383px;">Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-4-chloro-3-indolyl phosphate (BCIP)</td>
			<td>Sigma</td>
			<td style="width:137px;">N6639</td>
			<td>Protect this solution from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitroblue tetrazolium (NBT)</td>
			<td>Sigma</td>
			<td style="width:137px;">840W</td>
			<td>Protect this solution from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from Bovine Serum, purified fraction V (BSA)</td>
			<td>Sigma</td>
			<td style="width:137px;">A8806</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sheep Anti-digoxigenin-AP Fab Fragments</td>
			<td>Roche</td>
			<td align="right">1093274910</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516-500ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well cell culture plates</td>
			<td>Sarstedt</td>
			<td align="right" style="width:137px;">83.1835</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2</td>
			<td>ZIRC</td>
			<td style="width:137px;">Tg(mnx1:GFP)ml2</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean</td>
			<td>Aquarium Systems</td>
			<td style="width:137px;">N/A</td>
			<td style="width:383px;"></td>
		</tr>
	</tbody>
</table>

high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function

本文着重介绍整个安装的斑马鱼胚胎原位杂交（WISH）。无论是在组织中的分布和发育阶段基因表达的愿望技术有助于评估。议定书描述使用WISH斑马鱼胚胎，使用地高辛标记的反义RNA探针。通过在体外转录的基因已被克隆和线性化的模板，将地高辛联核苷酸探针所产生的。收获定义的发育阶段的胚胎绒毛膜特异性探针孵化前被删除。经过洗涤步骤，以除去多余的探针，胚胎用碱性磷酸酶偶联的抗地高辛抗体孵育。通过采用碱性磷酸酶的显色底物，特定基因的表达进行评估。根据不同的基因的表达水平，就可以完成整个过程需要2-3天。

每筐50个胚胎（每个基因/实验条件），该基因的表达模式，可以实现在一个实验中使用的协议。几乎所有的胚胎显示一个特定的基因表达模式相似。 原位杂交染色的代表性实例示于图3-6中 。 的正义和反义核糖探针，合成的cDNA对应于PC5.1，PC5.2 6，SCL/tal-1 7，GATA-1 8,9，FLK / kdrl的10，11嘘12 DLX2的fkd7 / FOXA1 13，14,15，胰岛素和胰?...

Watch this Scientific Journal Video about High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function at JoVE.com

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

在脊椎动物的发育和疾病的基因功能研究的模型，小热带鱼，斑马鱼已成为一种流行。靶基因表达的时间和空间可以由<em在原位</em&gt;杂交。我们改进后的协议允许低非特异性背景信号检测低丰度的成绩单。

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 ...

high-resolution-whole-mount-situ-hybridization-within-zebrafish

Research

JoVE Journal

Biology

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

22.1K 次观看.  Royal Victoria Hospital. 在脊椎动物的发育和疾病的基因功能研究的模型，小热带鱼，斑马鱼已成为一种流行。靶基因表达的时间和空间可以由

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

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

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

斑马鱼的胚胎显微注射来分析基因的功能

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

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 the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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 ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

放射性的原位杂交检测组织不同的基因表达模式

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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 ...

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

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

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

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

高解析度全山

摘要

探索更多视频

此视频中的章节