我想感谢许多本科学生在我的实验室工作，并为此作出了贡献，这些协议的演变。 NAT支持斯基德莫尔联盟网络，建立项目与美国国家科学基金会预先支付的补助金。

一，RNA 原位杂交技术在全山海洋软骨鱼类 1。胚胎的固定和准备<ol><li>滑板胚胎可以上演根据巴拉德7。用于检测基因表达的最优性阶段取决于感兴趣的组织。为了跟踪基因的表达在滑板消化道，阶段27-30是最佳7。 </li><li>解剖胚胎PBS，分离胚胎的卵黄囊。 </li><li>修正在30毫升新鲜配制的4％多聚甲醛（PFA）的50毫升锥形管中，在4℃用温和的摇摆。修正为24小时。 </li><li>洗净胚胎在PBT，两次15分钟，在室温下旋转。 RNA安装在原位的所有解决方案的食谱（ 表1）。 </li><li>脱水胚胎通过在甲醇系列的25％，50％和75％甲醇：PBT摆动在室温下洗涤​​。洗涤的持续时间取决于在舞台上的胚胎。对于的pre-neurulation上演胚胎，甲醇系列中的每个洗涤5分钟就足够了。对于胚胎以上的级30，洗涤可以持续长达30分钟。胚胎，以确定是否有足够的渗透和适应每次洗脸，保持直立管你的手。 ，如果胚胎沉管的底部，那么你就准备下洗。如果胚胎浮到溶液的顶部的，改变的溶液，并重复该特定的脱水工序。 </li><li>在100％甲醇洗两次，10分钟，轻轻摇摆。 </li><li>一旦脱水到100％甲醇，胚胎应储存在-20℃下至少24小时，然后再进行与该协议。胚已被成功地存储在-20℃下，用于RNA整体透最多到1年。 </li></ol> 2。合成的RNA探针<ol><li>你要检测的基因，其表达产生一个线性片段。这是可以做到通过PCR扩增或线性化的质粒与您的基因的兴趣。通过PCR扩增，选择引物的结合位点侧翼基因的插入和保留在扩增区域的RNA聚合酶结合位点。对于常用的质粒如pBS中或载体pGEM，M15正向和反向引物是理想的的。或者，线性化的质粒，通过用酶消化QIAGEN公司小量制备DNA的15微升，在该基因的5&#39;末端的裂解。凝胶提取，纯化使用适当的QIAQUICK工具包。 </li><li>反转录的RNA探针，用8微升的PCR产物作为模板，使用适当的RNA聚合酶的转录反应（ 见表2）的线性质粒或100 ng。 </li><li>孵育逆转录反应在37℃下2小时</li><li>要确认反应，运行1微升的1％琼脂糖/ TAE凝胶上的转录反应。运行“凝胶在100 mVolts的，直到染料的凝胶正好碰上。一根突出的乐队应该是可见的。的线性DNA模板片段可能是在更高的抽氢隐约可见LAR的重量。 </li><li>加入1μl的无RNA酶的DNA酶I至转录反应和孵育在37℃下持续15分钟。 </li><li>沉淀，在-20℃下添加100微升TE pH值为8，将10μl的4M氯化锂，加入300μl100％乙醇，并培育至少60分钟或过夜。 </li><li>自旋以14,000 rpm在4℃离心10分钟。 </li><li>洗净颗粒，用70％的乙醇和空气干燥。 </li><li>重悬在20微升蒸馏水中。RNA探针添加80μl的杂交缓冲液（杂交缓冲液中，必须预先温热至70℃）。该探针可以被存储在80％的杂交缓冲液中，在-20℃或更长的时间为几个月，在-80℃下</li></ol> 3。胚胎前处理和杂交<ol><li>清除胚存储从-20℃，在​​100％甲醇对于年轻的胚胎进行步骤（3.2）。对于阶段后上演胚胎（29/30），你可能会看到一个表皮层，体内的胚胎&#39;取消&#39;关闭。在显微镜下，小心地羞辱等最大限度地探头渗透表皮层。 </li><li>将胚胎放置在干净的玻璃闪烁瓶。再水合的胚胎中的上述反向甲醇系列：（75％，50％，25％的甲醇：PBT）为5-30分钟，在室温下通过轻摇每个解决方案。完整的补液的PBT洗两次，每次10分钟，轻轻摇晃。 </li><li>要删除的任何颜料，漂白剂的胚胎在PBT中的6％的过氧化氢在室温下1小时，摇摆。 </li><li>删除在PBT洗涤3次，过氧化氢，（在室温下，每次10分钟摇动）。 </li><li>治疗用蛋白酶K，以便渗透的探针在杂交的胚胎。蛋白酶K的量和治疗持续时间取决于您要检查的组织和胚胎的年龄。更多的浅表组织和年轻的胚胎需要较短少蛋白酶K，而深层组织和年龄较大的胚​​胎，需要较高的浓度和更长的治疗添e的完全通透探针杂交的胚胎。例如，要检测的Shh在肠道内胚层的阶段29溜冰鞋胚胎，30微克/毫升的蛋白酶K / PBT的是，在室温下孵育20分钟。 </li><li>快速冲洗胚胎在PBT洗去蛋白酶K </li><li>要停用蛋白酶K，4％PFA，PBT 0.2％的戊二醛20分钟，轻轻摇摆的后缀胚胎。 </li><li>清洗两次，每次10分钟，PBT。 </li><li><ol class="lalpha"><li>预杂交，预热杂交液中加入2-3毫升的胚胎，就足以覆盖他们。轻轻摇动在恒温水浴中，在70℃1小时。 </li><li>为了制备RNA探针杂交溶液，预热至70℃，10分钟，并在冰上冷却。 15-20微升RNA探针稀释至2毫升新鲜预热的杂交溶液中，以获得最终的浓度为0.5-1.0微克/毫升。删除前hybe，加入稀释的RNA探针对胚胎。胚胎应覆盖的解决方案，日的杂交解决方案，如果有必要的话。紧密安全的盖子，闪烁瓶。杂交的轻摇在一个加热的水浴中于70℃搅拌过夜。 </li></ol></li></ol> 4。发表杂交清洗和抗体杂交<ol><li>删除从在新鲜的闪烁小瓶或Eppendorf管中的胚胎和地方与探针杂交溶液。该探针可以被存储在-20℃，并在将来重用。杂交后清洗，地方胚胎在Netwe​​ll的坐在6孔培养板中。 </li><li>每个预先温热解＃1，摇摆在70℃30分钟，洗涤3次</li><li>每个预先温热解＃2，摇摆在70℃30分钟，洗涤3次</li><li>洗涤3次，每次5分钟在TBST中，在室温下摇动。 </li><li>块的前10％热灭活的羊血清在TBST中1小时，在室温下摇动。 </li><li>转移的胚胎从Netwe​​ll到一个新的玻璃闪烁瓶。抗-DIGFab段（1:5,000），在1％热灭活的羊血清/ TBST中胚胎。摇滚在4°C过夜</li></ol> 5。后抗体杂交洗<ol><li>删除抗体和丢弃。地点胚胎背部到Netwe​​ll的清洗。 </li><li>洗涤3次，每次5分钟在TBST中，在室温下摇动</li><li>洗净至少6次，每次1小时，在室温下摇动。 </li><li>洗净，摇摆于4℃过夜，TBST作为后抗体洗涤有助于减少背景染色，最大限度地提高洗涤的次数和持续时间是最好的。的胚胎是经常在TBST洗涤，在4°C，在上周末。 </li></ol> 6。检测探头<ol><li>新鲜NTMT解决方案和过滤消毒。 </li><li>丢弃的TBST洗涤液和地方胚胎在干净的玻璃闪烁瓶。洗净胚胎3次每次10分钟，在室温下，摆动的新鲜NTMT。 </li><li>删除NTMT洗净，加入反应混合物的1×NBT / BCIP显在1×NTMT。由于这些反应物是光敏感的，覆盖在箔和岩石的闪烁瓶中，在室温下。 </li><li>监控显色反应，每15分钟，直到所需的基因表达清晰可见。强探头可能需要10分钟发展。弱探头可能需要1-2小时。不要让过长或将开始出现的背景染色（全胚胎开始转暗紫色或棕色）的反应。 </li><li>洗2次，每次10分钟在室温下，摆动在PBT。 </li><li> Postfix的胚胎在4％多聚甲醛中，在室温下的1小时的0.1％戊二醛。胚胎可以无限期存储在固色剂，在4°C </li><li>可视化胚胎解剖体视显微镜。为了产生一个淡蓝色背景的图片，产地胚胎在培养皿中预硬化的1％琼脂糖/ PBS。 </li></ol> 7。一，RNA 原位杂交技术在全山海洋软骨鱼类的代表性成果 RNA安装在原位的描绘表达的刺猬 （ 嘘），HOXA13在滑板胚胎的， 如图1所示。脊索和内脏内胚层在高等脊椎动物中发现的 Shh的表达，这种表达模式是保守的冰鞋（ 图1a）8,9。海洋软骨鱼类有独特的方法，渗透调节，使用的直肠腺分泌盐。，HOXA13表达是在发展中国家的直肠腺（ 图1b）10。HOXA13基因的产品在图案中的作用的直肠腺仍是未知的。 II。石蜡包埋，切片软骨鱼类组织 1。收获的组织和准备<ol><li>解剖所需的组织和固定在10％福尔马林浸泡24-48小时，在室温下摇动。 4％多聚甲醛（PFA），也可以用来作为一种固定（见讨论）。 </li><li>通过在PBS中洗涤3次，每次30分钟，洗净，满分固定液。根据组织的大小，洗涤时间可以降低到10分钟，或增加至1小时。 </li><li>由在室温下在25％，50％和75％乙醇的乙醇系列：PBS摆动洗涤脱水胚胎。同样，每次洗涤的时间将依赖于组织的大小。 0.5厘米3组织，每次洗涤15分钟孵育。增加较大块的时间。如果组织的长期存储需要，洗一个额外的2倍，在75％乙醇中，并存储在-20℃下长达一年。否则，请继续下一个步骤。 </li><li>另外，脱水，在100％乙醇洗涤3次，每次15分钟，摇摆。 </li></ol> 2。石蜡包埋，切片<ol><li>明确组织在二甲苯洗涤2次，每5分钟与螺丝帽盖在玻璃闪烁瓶。二甲苯会使组织变脆，所以不要超过10分钟，共为是住户开支统计调查。组织应该看看半透明的，当你拿着它到光。如果组织仍然是非常不透明洗两次后，在二甲苯中做额外的清洗5分钟，然后进行协议。只有处理好与手套，在通风橱内的二甲苯。 </li><li>删除二甲苯和闪烁瓶的2/3熔化的石蜡填充。孵育在60℃的烘箱中30分钟。当把石蜡，你会发现到小瓶的石蜡凝固沿两侧的小瓶。样品瓶放置在烤箱中，并允许石蜡重新溶解了几分钟。从烤箱里拿出瓶和解决方案的快速漩涡混合和替换烤箱其余的潜伏期。 </li><li>重复石蜡孵育30分钟两次。 </li><li>石蜡两次一个小时的孵育。更改石蜡，最后一次在60°C的烘箱中孵育过夜。 </li><li>翌日，放置2-3小时中的组织在真空下加热的石蜡浴室和地点。这将有利于石蜡渗透到组织中，并除去任何气泡。对于某些类型的薄的组织，可能并不需要的真空步骤，但它是必要的整个胚胎或消化道和与腔室的其他器官。 </li><li>真空后促进组织浸润，地点组织，在金属模具用熔化的石蜡，和冷却在冰板。发表在冰上或4℃过夜，然后试图从模具中取出组织。组织的石蜡块可以无限期储存在4℃下</li><li>地点石蜡切片机，切块到8微米的部分。段应漂浮在水浴中加热至48°C，并安装到和玻璃的Superfrost *加幻灯片。 </li><li>干片，在37℃的烘箱中过夜。 </li><li>在4°C，直到需要的存储部分。 </li></ol> III。阿尔辛蓝/核固红染色软骨鱼组织 1。阿尔辛蓝染色粘蛋白<ol><li>地点滑动的部分朝上，幻灯片上的回暖。熔体45分钟。 </li><li>地方滑入一个幻灯片持有人。所有的相关的解决方案都包含在一个通风橱内的浴缸。通过将他们从一个浴缸的解决方案到另一个幻灯片进行清洗。确保在浴缸的解决方案，包括幻灯片，在幻灯片上的组织被完全淹没。溶解石蜡洗涤幻灯片在二甲苯中的2次，每次5分钟。 </li><li>洗净幻灯片两次，每次1分钟，用100％乙醇。 </li><li>再水合的乙醇系列中的幻灯片（75％，50％，25％乙醇），在各溶液中洗涤1分钟。在DH 2 O洗1分钟。 </li><li>染色法在15分钟爱茜蓝溶液，pH为2.5。 </li><li>开发运行3分钟自来水洗涤染色。一旦浸在蒸馏水中。 </li><li>染液核固红染液溶液中10分钟。 </li><li>在3分钟自来水冲洗，浸泡在蒸馏水2 O一次。 </li><li>脱氢DRATE系列的乙醇（25％，50％，75％，100％，100％）为20每次浸，或每次1 min。在二甲苯中洗2次，每次5分钟。安装与DPX安装介质和盖玻片。允许安装在通风橱内48小时前操纵幻灯片中干燥变硬。 </li></ol>

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A., Simeone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+rudimentary+colon+in+the+elasmobranch,+Leucoraja+erinacea.">Evidence of a rudimentary colon in the elasmobranch, Leucoraja erinacea.</a> Dev. Genes Evol. 222, 237-243 (2012).</li><li>Filipe, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins+in+the+human+gastrointestinal+epithelium:+a+review.">Mucins in the human gastrointestinal epithelium: a review.</a> Invest. Cell Pathol. 2, 195-216 (1979).</li><li>Corfield, A. P., Wagner, S. A., Clamp, J. R., Kriaris, M. S., Hoskins, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucin+degradation+in+the+human+colon:+production+of+sialidase,+sialate+O-acetylesterase,+N-acetylneuraminate+lyase,+arylesterase,+and+glycosulfatase+activities+by+strains+of+fecal+bacteria.">Mucin degradation in the human colon: production of sialidase, sialate O-acetylesterase, N-acetylneuraminate lyase, arylesterase, and glycosulfatase activities by strains of fecal bacteria.</a> Infect. Immun. 60, 3971-3978 (1992).</li><li>Reid, B. J., 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=Flow-cytometric+and+histological+progression+to+malignancy+in+Barrett's+esophagus:+prospective+endoscopic+surveillance+of+a+cohort.">Flow-cytometric and histological progression to malignancy in Barrett's esophagus: prospective endoscopic surveillance of a cohort.</a> Gastroenterology. 102, 1212-1219 (1992).</li><li>Mowry, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+staining+of+pancreatic+beta-cell+granules.+Evolution+and+present+status.">Selective staining of pancreatic beta-cell granules. Evolution and present status.</a> Arch. Pathol. Lab Med. 107, 464-468 (1983).</li><li>Roberts, D. J., Smith, D. M., Goff, D. J., Tabin, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+signaling+during+the+regionalization+of+the+chick+gut.">Epithelial-mesenchymal signaling during the regionalization of the chick gut.</a> Development. 125, 2791-2801 (1998).</li></ol>

我什么都没有透露。

的协议是典型的监测基因表达的方法，并确定分化的细胞类型，并已被改编为海洋软骨鱼类。这些协议可能需要进一步的修改，以适应不同的软骨鱼物种。 最常见的关切RNA整装原位的是RNase的污染的风险，从而降解的RNA探针和内源性消息。需要考虑两个方面：合成的核糖体探针和杂交胚胎邮件，和一般的技术处理RNA。在一般情况下，由核糖核酸酶的污染可避免从容器未...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>10 x transcription buffer</td>
 <td>Roche</td>
 <td>11-465-384-001</td>
 <td></td>
 </tr>
 <tr>
 <td>DIG-RNA labeling mix</td>
 <td>Roche</td>
 <td>11-277-073-910</td>
 <td></td>
 </tr>
 <tr>
 <td>RNAse inhibitor</td>
 <td>Roche</td>
 <td>03-335-399-001</td>
 <td></td>
 </tr>
 <tr>
 <td>RNA polymerase - SP6</td>
 <td>Roche</td>
 <td>10-810-274-001</td>
 <td></td>
 </tr>
 <tr>
 <td>DNAseI, RNAse-free </td>
 <td>Roche</td>
 <td>10-776-785-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Yeast RNA</td>
 <td>Invitrogen</td>
 <td>15401-029</td>
 <td></td>
 </tr>
 <tr>
 <td>CHAPS</td>
 <td>EMD-Millipore</td>
 <td>220201</td>
 <td></td>
 </tr>
 <tr>
 <td>heparin</td>
 <td>Sigma-Aldrich</td>
 <td>H4784</td>
 <td></td>
 </tr>
 <tr>
 <td>DEPC (diethyl pyrocarbonate)</td>
 <td>Research Organics</td>
 <td>2106D</td>
 <td></td>
 </tr>
 <tr>
 <td>Moria Perforated Spoon</td>
 <td>Fine Science Tools</td>
 <td>10370-17</td>
 <td></td>
 </tr>
 <tr>
 <td>Netwell inserts</td>
 <td>Electron Microscopy Sciences</td>
 <td>64713-00</td>
 <td>Netwells for use in 6-well tissue culture dishes</td>
 </tr>
 <tr>
 <td>6-well tissue culture plate</td>
 <td>Corning</td>
 <td>3516</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass scintillation vials with screw-cap lids</td>
 <td>Weaton Science Products</td>
 <td>986540</td>
 <td></td>
 </tr>
 <tr>
 <td>formamide</td>
 <td>Fisher</td>
 <td>BP227500</td>
 <td></td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Invitrogen</td>
 <td>59895 (AM2542)</td>
 <td></td>
 </tr>
 <tr>
 <td>NBT</td>
 <td></td>
 <td>11585029001</td>
 <td></td>
 </tr>
 <tr>
 <td>BCIP</td>
 <td>Roche</td>
 <td>11585002001</td>
 <td></td>
 </tr>
 <tr>
 <td>Hydrogen peroxide, 30%</td>
 <td>EMD</td>
 <td>HX0635-1</td>
 <td></td>
 </tr>
 <tr>
 <td>Sheep serum</td>
 <td>VWR</td>
 <td>101301-478</td>
 <td></td>
 </tr>
 <tr>
 <td>glutaraldehyde</td>
 <td>Sigma-Aldrich</td>
 <td>G5882</td>
 <td></td>
 </tr>
 <tr>
 <td>tRNA</td>
 <td>Roche</td>
 <td>10-109-541-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-DIG Fab Fragments</td>
 <td>Roche</td>
 <td>1137-6623</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 3. Reagents and equipment for RNA whole mount in situ's.</td>
 </tr>
 <tr>
 <td>1% Alcian Blue 8GS, pH 2.5</td>
 <td>Electron Microscopy Sciences</td>
 <td>26323-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Nuclear Fast Red</td>
 <td>Electron Microscopy Sciences</td>
 <td>26078-05</td>
 <td></td>
 </tr>
 <tr>
 <td>DPX Mountant</td>
 <td>Electron Microscopy Sciences</td>
 <td>13510</td>
 <td></td>
 </tr>
 <tr>
 <td>Paraffin (Paraplast X-tra)</td>
 <td>McCormick Scientific</td>
 <td>39503002</td>
 <td></td>
 </tr>
 <tr>
 <td>10% Formalin, NBF</td>
 <td>VWR</td>
 <td>95042-908</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass scintillation vials with screw-cap lids</td>
 <td>Weaton Science Products</td>
 <td>986540</td>
 <td></td>
 </tr>
 <tr>
 <td>Stainless steal base molds</td>
 <td>Tissue-Tek</td>
 <td>4161-4165</td>
 <td>Multiple sizes available.</td>
 </tr>
 <tr>
 <td>Cassettes </td>
 <td>Tissue-Tek</td>
 <td>4170</td>
 <td></td>
 </tr>
 <tr>
 <td>Slide warmer</td>
 <td>Fisher-Scientific</td>
 <td>12-594Q</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue Embedder</td>
 <td>Leica Microsystems</td>
 <td>EG1160</td>
 <td></td>
 </tr>
 <tr>
 <td>Microtome, rotary</td>
 <td>Leica Microsystems</td>
 <td>RM2235</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue-Tek Slide Staining Set</td>
 <td>Electron Microscopy Sciences</td>
 <td>62540-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue-Tek 24-Slide Holder</td>
 <td>Electron Microscopy Sciences</td>
 <td>62543-06</td>
 <td></td>
 </tr>
 <tr>
 <td>Superfrost*Plus slides</td>
 <td>Fisherbrand</td>
 <td>12-550-17</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Table 4. Reagents and equipment for Alcian Blue stain.</td>
 </tr>
 </tbody>
</table>

rna in situ hybridization in whole mount embryos and cell histology adapted for marine elasmobranchs

海洋软骨鱼类生物医学和基因组学研究的动物模型的价值，因为他们是最原始的脊椎动物适应性免疫，并有独特的渗透调节机制为1-3。作为最原始的生活有颚脊椎动物与配对的附属物，软骨鱼类进化是一个重要的模型，特别是在进化和发展的研究。海洋软骨鱼类也被用于研究水生生物毒理学和逆境生理关系，气候变化4。因此，发展和适应的方法是必要的，以促进和扩大利用这些原始的脊椎动物多个生物学科。在这里，我成功适应的RNA整个安装在原位杂交和组织学技术在软骨鱼类的基因表达和细胞组织学研究。 监测基因表达的特点是发育生物学工具学家，被广泛用于研究发育过程。 RNA 原位杂交，使发育中的胚胎组织中的特定基因转录的可视化和本地化。能够洞察发育过程中细胞命运决定基因可以控制基因的表达模式的消息。通过比较在不同发育阶段的基因的表达模式，洞察可以得到如何在开发过程中的作用的一个基因的改变。 虽然整个安装在原位的提 ​​供了一种基因表达的本地化组织，组织学技术允许分化的细胞类型和组织的鉴定。组织学污渍有不同的功能。一般污渍是用来突出，例如细胞形态的细胞核和细胞质对于一般性的染色，苏木精和曙红。其他污渍可以突出显示特定的细胞类型。例如，阿辛蓝染色本文报道的是一种广泛使用的阳离子染色，以确定mucosaccharides。阿尔新蓝染色，消化道可以识别分布杯状细胞产生mucosaccharides的的。短肽mucosaccharide成分变化区分杯状细胞的功能，消化道内的6。同时通过RNA安装在原位的组织化学方法，细胞命运的决定可以被链接到特定基因的表达。 虽然RNA 原位的组织化学的研究人员，他们的适应和利用海洋软骨鱼类被广泛应用于满足有限的，不同程度的成功。在这里，我提出的软骨鱼类和开发，在我的实验室定期使用的协议。虽然可能需要进一步的变形例中的RNA 原位的杂交方法，以适应不同的物种，这里描述的协议p海洋软骨鱼类的使用要适应他们的科学调查的研究人员开展交流一个有力抓手。

在不同地区的L.阿尔新蓝染色的例子在图2中所示erinacea消化道。酸的粘蛋白含有globlet细胞是清晰可见整个消化道的阿辛蓝染色。酸粘蛋白的分布不同，在消化道的不同区域，从而反映出在功能上的差异。酸性的粘蛋白在螺旋小肠和泄殖腔稀疏产生的，而在远侧小肠（比较图2a和 2c与2b中 ）检测到高浓度的酸粘蛋白。 <table border="1" fo:keep-together....

Watch this Scientific Journal Video about RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs at JoVE.com

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

通过RNA整装相结合的方法<em在原位</em&gt;杂交和组织学，基因表达可在胚胎发育与细胞命运的决定。这些方法已经适应了海洋软骨鱼类和方便的使用这些动物作为生物医学，毒物学和比较研究的模式生物。

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 ...

rna-situ-hybridization-whole-mount-embryos-cell-histology-adapted-for

Research

JoVE Journal

Biology

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

12.6K 次观看.  Union College. 通过RNA整装相结合的方法

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

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 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 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

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 ...

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

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

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

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

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

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

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

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

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

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解析度全山<em&gt;在原位</em杂交斑马鱼胚胎内，研究基因表达和功能

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...