作者要感谢所有的贾维斯实验室成员多年来的改进协议。

1。组织准备<ol><li>收获的新鲜组织。对于禽流感的胚胎，小心地打开鸡蛋和一碟与1xPBS，两次清理胚胎。对于成人的大脑，迅速取出大脑，轻轻地用1×PBS。 </li><li>嵌入到一个嵌入式模具华侨城组织TEK的胚胎或成人的大脑，定向组织切片需要，并迅速冻结成乙醇和干冰的混合物，小心不要让块内的混合物块。 </li><li>切片冷冻样品在低温恒温器到10-12微米厚的部分。大脑和胚胎组织，最佳的切割温带至-20°C -18°C的范围内是</li><li>超级加玻片上挂载的冰冻切片。 </li><li>在幻灯片中的部分储存在-80°C </li></ol> 2。放射性Riboprobes代（使用您的机构辐射安全程序） <ol><li>生成一个纯净的线性的DN你感兴趣的cDNA通过限制消化酶的RNA聚合酶从质粒DNA或RNA聚合酶启动子的结合位点插入PCR子网站包围的片段模板。它也有可能产生PCR片段与RNA聚合酶启动子3&#39;和5&#39;PCR引物的一部分。凝胶净化与GENECLEAN凝胶纯化试剂盒的限制或PCR产生的碎片。 </li><li>到0.5毫升的Eppendorf管中，加入0.5-1微克纯化的线性DNA模板，转录缓冲，数码地面电视，RNasin，AGC核苷酸混合溶液，核糖核酸酶自由水带来的音量所需的金额。然后加第35 UTP和适当的RNA聚合酶，使无论是反义或感riboprobes。 </li><li>为1小时37℃水浴中孵育的混合物。添加另取RNA聚合酶和孵育另1个小时。 </li><li>添加3M醋酸钠溶液（总体积的0.1倍）和100％乙醇（2.5 FOLD总量）到Eppendorf管沉淀合成核糖核酸riboprobe。 </li><li>孵育在干冰或-80°C下15分钟或3个多小时，以协助降水。 </li><li>颗粒的RNA离心在4°C和30分钟在一个表上的Eppendorf离心机15000转。 </li><li>去除上清，用70％乙醇沉淀，挖掘用手指混合颗粒。 </li><li>颗粒的RNA再次离心4°C和30分钟15000转。 </li><li>由pipeting完全去除上清，然后加入40μL杂交液。拌匀由pipeting和。 </li><li>投入1微升的溶液3毫升的安全解决解决方案，在闪烁瓶中，拌匀，并测量在闪烁计数器计数。 </li><li>放置在-20°C的一个星期内使用的riboprobe Eppendorf公司。 </li></ol> 3。组织处理<ol><li>在引擎盖，准备新鲜的PBS缓冲4％paraformaldehy约3-5°C以上室温的解决方案。地方幻灯片从-80°C储存在干冰上的金属架，引擎盖下的工作空间进行，然后将其放置到机架的4％多聚甲醛溶液，在室温下5分钟的孵育。 </li><li>在1×PBS洗3次（每浸1秒左右）约15骤降。 </li><li>在引擎盖上，使乙酰缓冲区和大力摇晃10秒内。立即倒入一个包含幻灯片机架托盘，孵育10分钟，以减少背景的riboprobe的结合。 </li><li>在2×SSPE的15骤降冲洗3次。 </li><li> 70％，95％和100％乙醇2分钟，在每一步串行脱水（没有在引擎盖）。 </li><li>引擎盖下干燥至少10-15分钟的幻灯片。 </li></ol> 4。杂交<ol><li>计算的riboprobe量（0.5-1×10 6每幻灯片CPM）和杂交液（100μL每张幻灯片）所需的所有幻灯片。 prewaRM的riboprobe杂交组合，以65°C 5分钟变性的riboprobe的。 </li><li>吸取100μL杂交riboprobe解决方案在整个幻灯片线，使用盖玻片遍布均匀的组织和盖玻片。地方滑出水平，面对直立的金属架和地方机架到至少4小时65℃的油浴和最多16个小时，慢慢直立。油创建周围的盖玻片密封不透气。 </li><li>从油浴金属架和机架用纸巾擦拭周围多余的油。 </li><li>从覆盖的幻灯片和金属机架中含有氯仿，两次的玻璃或金属盘洗掉油。可以作为未来实验的第一第二氯仿洗。 </li><li>转移到一个托盘机架覆盖幻灯片在0.1％β-巯基乙醇+ 2×SSPE的解决方案和移动数骤降和放松盖玻片和删除多余的杂交溶液溶解TION。 </li><li>转让机架新鲜的0.1％β-巯基乙醇+ 2 x SSPE的解决方案中涉及的幻灯片，然后取出盖玻片与解决方案，以防止刮伤组织切片中的核糖核酸酶自由钳。在一个托盘水平滑动机架持有人转移到新鲜机架裸露的幻灯片，在0.1％，2×SSPE的β-巯基乙醇溶液。 </li><li>只孵育新鲜的0.1％β-巯基乙醇的幻灯片机架+ 2 x SSPE的溶液在室温1小时，以去除多余的绑定RNA探针。弃掉此以前的水清洗解决方案，以及盖玻片，作为放射性废物。 </li><li> 1预热2X SSPE的解决方案，在65°C间转移的机架用幻灯片，添加β-巯基乙醇的0.1％浓度的最后，并在65°C孵育1小时。放射性废物丢弃。 </li><li>转移和孵化机架预热0.1 x SPPE幻灯片两次在65°C间，每次30分钟。放射性物质的量中删除这一步是非常小的，这一步后不再被认为是过度的放射性废物。 </li><li>脱水70％的机架和幻灯片，95％和100％乙醇各2分钟。 </li><li>在引擎盖晾干至少30分钟的幻灯片。 </li></ol> 5。放射性信号的可视化<ol><li>将干片成片盒，并在黑暗的房间里，放置在幻灯片的X射线胶片（柯达百玛士议员电影），并关闭磁带。确保幻灯片所面临的X射线胶片的药膜。 〜1-7天预计大量的成绩单上取决于暴露的幻灯片。 </li><li>制定标准显影液和定影液的X光片。杂交信号显示为黑色电影（暴露在银颗粒乳剂）（ 图1）。 </li><li><ol class="lalpha"><li> （可选）要确定细胞的决议和组织上的信号，幻灯片，需要蘸感光乳剂和counterstained。如果你想最终甲酚紫染液，然后delipidize由二甲苯在室温孵育5分钟，两次，1分钟，在100％，100％，95％，95％，70％，50％的乙醇水化的部分然后在去离子水。如果你不打算染色用染料，不需要delipidization，然后delipidization的是没有必要的。至少2-3个小时的引擎盖下的干片。 </li><li>在黑暗的房间使用一个安全的光，舀出足够的柯达非关税壁垒乳液，以支付一半的幻灯片纵向（即组织）在玻璃容器中浸泡，然后融化在42°C的水浴20-30分钟。然后用蒸馏水以1:1的比例稀释。乳液的水平，现在应该包括它蘸的幻灯片时，幻灯片上的所有部分。如果你有很多幻灯片，你可能需要准备额外的乳液。 </li></ol></li><li>到摊薄乳液浸在42℃水浴中，在一个封闭的光密封的容器中干浸幻灯片overnig幻灯片在漆黑的房间里，或在2-3小时的烤箱，在37°C，关灯，HT。 </li><li>转移到机架插槽幻灯片中含有干燥器的黑盒子，幻灯片小心，不能互相接触，从而创造文物。用黑色电工胶带密封的箱子的边缘，慢慢来防止静电引起的光火花，然后包裹在铝箔盒。存储的框，在4°C间从数天到数周（1天X射线胶片上的信号是类似乳液下5天）。 </li><li>温暖的幻灯片框1小时至室温。 </li><li>在暗室中，机架（或在一个金属架的地方幻灯片与幻灯片中删除，如果使用滑槽框）从箱子他们在柯达的D-19的开发和发展在16°C为3.5分钟。 </li><li> 1分钟，洗净，在室温下的自来水发达的幻灯片。 </li><li>孵育在19°C两次，每次6分钟的幻灯片在定影液。灯可以开启期间第二定影液孵化。 </li&gt; <li>运行在室温下至少30分钟的水冲洗的幻灯片，并从幻灯片的背面刮的乳液，而“湿”用刀片，以防止刮伤玻璃。 </li><li> 5分钟与自来水中的0.3％的甲酚紫染色组织。 </li><li>在新鲜的自来水洗〜15逢低额外甲酚紫的解决方案。 </li><li>脱水：50％〜15逢低酒精溶液中每个幻灯片，70％，95％，95％，100％和100％的乙醇。 </li><li>在室温下5分钟孵育两次在二甲苯中的幻灯片。 </li><li>盖玻片用幻灯片和干燥过夜（&gt; 16小时）在引擎盖覆盖幻灯片;将需要数天前的胶水是不够坚固，进一步清理幻灯片Permount中等。 </li></ol> 6。产生暗场彩色图像<ol><li>如果必要，进一步清理多余乳液的润湿水，并用刀片刮，无组织（非coversliped的侧）后面的幻灯片。 </li> &lt;李&gt; 80％的乙醇溶液冲洗幻灯片，轻轻擦拭1-2次，去除碎片和尘埃。 </li><li>采取暗场或明照明下的照片。 6.2和6.3的步骤可能要重复2-3次，以获得一个很好的无尘粒，很容易在解剖显微镜下看到在暗场图像。 </li></ol> 7。代表结果 “第35放射性探针杂交的组织切片原位杂交结果上观看的两个主要途径：1）X光片被放置在幻灯片或2）乳液涂幻灯片。第三种方法是使用phosphorimager屏幕放置在幻灯片，但我们尚未与本办法的决议感到满意。 X光片提供了一个快速的结果和分析的杂交整体状况。 X-射线胶片数据还揭示了广泛的解剖分辨率和可用于定量分析产品是10。禽流胚胎后期头杂交的FOXP1和CoupTF2基因表达的反义探针X射线胶片图像的例子是在图1A和B，这两种基因是在特定的大脑细分高度丰富。一个质量好的X光片的结果应该是夏普（不模糊）和高信号背景比。一个朦胧的形象，可能是由于X-射线胶片与杂交组织玻片之间的不平衡接触。 对于乳液浸幻灯片，乳液含有光敏感的银组织作为apposed X射线胶片的塑料涂层的盐。在发展中国家，第35曝光的银盐转换为金属银颗粒，很像在X光片，。然而，银矿床是直接在细胞的基因表达，可以在显微镜下观察和测量定性可见。金属银颗粒阻止直接光线通过明观下的黑点出现。甲酚紫染液出现紫色（ 图3A，3C，图4）。在暗场，银颗粒反射光线从侧面出现白点（ 图1C，1D，3B，3D）。在这种情况下，甲酚紫染色出现红色。在明，杂交信号更容易查看下在蜂窝分辨率高倍率，而在暗场，此外，杂交信号，可以在整个组织中的低倍率观看。的暗场的看法是，我们通常用它来显示整体的基因表达模式的方法。然而，相对快速的结果，从X光片获得，乳液浸幻灯片的需要更长的时间（几个星期），并获得背景更为敏感。 强大的背景有四种常见的来源：1）所有的X光片的背景通常做与开发商或定影液，或部分的曝光胶卷的问题; 2）玻片上的背景通常是由于洗涤或玻片的准备，如从商业来源或自制幻灯片不当silination问题; 3）乳液部分由于缺乏仔细杂交后的洗涤步骤，杂交温度过低，质量较差的杂交液，洗碗的多聚甲醛污染造成探针永久交联的背景曝光和发展的背景下，以及4）的组织，riboprobe退化，导致小分子非特异性标记组织，无效的DTT或β-巯基乙醇在交叉连接二硫化债券的S 35-RNA探针组织，并等待对乙酰太长。醋酸酐和三乙醇胺混合秒内乙酰化解决方案的幻灯片，这是至关重要的。如果几分钟传递withouţ的解决方案添加到幻灯片，然后乙酰基不会被有效去除，然后绑定到非特异性的RNA。其他因素包括杂交超过20小时，这可能会产生太强烈的信号，并在幻灯片上，它封存在幻灯片上的杂交液，在清洗剂，导致组织的放射性点多余的油滴和幻灯片给黑暗的背景信号。如果有很多幻灯片（超过100片），加3 路氯仿洗或更改氯仿洗涤，其余的幻灯片上，以防止多余的油颗粒。组织处理不慎，即不冻结速度不够快（解剖后5-10分钟内）或解冻和重新冻结，也增加背景由于mRNA降解。小心，不要误解为背景的过度曝光。 有关乳液浸幻灯片的背景是可能的，因为它是高光敏感，并且需要在黑暗中长时间曝光。 COM<html>周一背景的问题包括显影液和定影液温度太高。当温度超过19°C间，接近或高于室温温暖，得到更多的银粮背景。泄漏到一个暗室的光线照射到水平低会导致乳液背景。不洗定影液足够长的时间（至少30分钟，自来水），将离开定影液，然后用甲酚紫反应产生整个乳液褐色的沉淀。然而，如果幻灯片甲酚紫染色前固定后超过90分钟洗涤水不再，这可能会导致乳液变得松散，染色不佳的部分。如果没有足够的时间在30-90分钟窗口染色后固定和洗涤后的30分钟洗净，幻灯片，干隔夜的幻灯片，并进行甲酚紫染色第二天。一般来说，大多数的基因有特定的基因表达模式，而背景信号更均匀。 e_content“&gt;折叠组织基因表达的结果可能误导X射线胶片暗信号，导致一个地区，要确定，如果组织被折叠，非彩色部分在明下暗场或甲酚紫染色切片检查。甲酚紫counterstaining提供一个更好的方法来检查的组织条件。 为探针特异性更好的诠释，应适用于控制感探头在几个相邻的部分。最有意义的探头不显示信号，但有些，当他们这样做，我们发现，它比反义信号往往是不同的。我们认为，这可能与反义合成或其他基因的基因组上的反义链。我们提出了一个例子，PAX6，反义探针（ 图2A和B）。反义链沿脑室区的前脑，小脑，如预期般的眼睛（ 图2A）标签，但意义上揭示LA贝林在视网膜色素层（ 图2B）。 探头尺寸，我们使用cDNA探针在300-5000个基点范围内的任何地方。探测不到300个基点的工作，但信号通常较弱。我们还没有尝试过探头超过5000个基点大。最好是从同一物种的组织使用的探针，如果可能的话。如果没有，我们交叉杂交探针对其他物种的部分路段和降低杂交和洗涤温度在3-5°C间基础上的同源性，如果已知增量。如果不知道，然后我们进行试验和错误的杂交和洗涤温度。如果温度降低过多，cDNA探针可以交叉杂交与其他基因跨物种或相似的序列，在11种相同。在实践中，我们发现的cDNA〜95％相同或更大的组织中的mRNA的目标，严格的杂交和洗涤温度（65℃）条件下工作得很好。对于序列的响〜50范围内，可能需要降低到60°C〜85至94％相同，杂交和洗涤温度的ES 在大多数的步骤中使用的金属架的原因是使用氯仿洗涤和二甲苯。这两种有机物融化多种塑料。玻璃和塑料的某些类型的抗这些有机物。但是，玻璃很容易打破，将融化长期间接触有机物和一些塑料制品，起初有抗药性。 <img src="/files/ftp_upload/3764/3764fig1.jpg" alt="图1"/> 图1。放射自显影原位杂交图像，从X光片和乳液浸幻灯片。 （AB）的X射线胶片图​​像矢状斑胸草雀鸣鸟在胚胎第10天（一）FOXP1或（B）与明照明CoupTF2，在解剖显微镜下，杂交反义riboprobes整个头部部分。黑色，暴露谷物显示表达明确的电影离子。比例尺= 500微米。 （CD）的乳液蘸矢状斑胸草雀的整个头部部分的幻灯片图像，在孵化后第6天与义riboprobes杂交（三）FOXP1（四）CoupTF2解剖显微镜下，用暗视野照明。白，银颗粒暴露在上面显示的mRNA表达的组织乳液。红，甲酚紫染色。比例尺= 200微米。所有图片，是左边的喙嘴。一天，浸幻灯片​​3天暴露在X-射线胶片图​​像。 FOXP1探头是178个基点至1544年至1711年BP的m​​RNA; CoupTF2是545个基点至1-545 bp的部分基因。可以看出，内脑FOXP1表达丰富的mesopallium（男），纹状体（ST）和背侧丘脑（DT），而CoupTF2丰富的nidopallium（N）arcopallium（一），腹侧丘脑。暴露类型和年龄之间的表达有一致性。与浸幻灯片，但是，更高分辨率的标签被看作，三维组织界限，分支机构直接确定。这些和其他所有的文件显示图像是使用标准的65°C的紧缩杂交部分。 <img src="/files/ftp_upload/3764/3764fig2.jpg" alt="图2"/> 图2。比较反义和正义的标签显示不同的图案。 （一）反义链的PAX6表达在大脑中，尤其是脑室区（白色箭头）。显示的是整个头部矢状斑胸草雀在胚胎第12天的片X光片显影。 （二）相邻节义链的PAX6杂交揭示整个胚胎头没有背景表达，但明显表达反义链（黑色箭头）在视网膜色素层。虚线表示的整个大脑的轮廓。 ：小脑。 <img src="/files/ftp_upload/3764/3764fig3.jpg" alt="图3"/> <st<html>荣&gt;图3。乳液浸幻灯片下旬萌芽阶段下的明场和暗场的意见，从斑胸草雀脑基因表达的原位信号。 （一）明场图像D1B表达在胚胎10天，从正常曝光的乳液。标签（黑），勉强可以看到在这个倍率。探头是625个基点至1-625 bp的部分基因。 （二）同第（一），但倍率切换视图显示标签的暗场（白）在纹状体（ST）和丘脑（TH）。 （三）明场图像Slit3表达在胚胎第12天从曝光过度乳化。标签（黑），可以很容易地看到。探头是779个基点，以1243年至2021年的mRNA部分。 （b）相同的部分，在（A）的放大倍率切换到暗场视图显示标签（白）在脊髓（SC），明的形象相匹配。延髓是面向左侧。 CB：小脑。 / 3764/3764fig4.jpg“ALT =”图4“/&gt; 图4。银粒在细胞水平上的决议。显示FOXP1在斑胸草雀脑mRNA的银颗粒（黑点），上述细胞在不同的大脑区域和年龄（甲酚紫）相比，低功耗影像图1A和1C的标签。 （一）对单个细胞的高丰度表达（黑色箭头）在成年斑胸草雀mesopallium。 （二）对单个细胞在同一节相邻nidopallium（黑色箭头），低丰度表达。 （C）最高FOXP1多细胞mRNA的表达在胚胎12天mesopallium（黑色箭头）。 （四）对细胞的低丰度表达（黑色箭头）在同一节相邻nidopallium。例如细胞与黄线盘旋。胚胎干细胞（C和D）是体积更小，更紧凑与成年细胞（A和B）。以来，有一些细胞和乳液之间的空间，面积暴露SI从S 35探针的的绿儿颗粒略高于胞体面积大。比例尺= 10微米。

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributions+of+two+gonadotropin-releasing+hormone+receptor+types+in+a+cichlid+fish+suggest+functional+specialization.">Distributions of two gonadotropin-releasing hormone receptor types in a cichlid fish suggest functional specialization.</a> J. Comp. Neurol. 495, 314-323 (2006).</li></ol>

我们什么都没有透露。

放射性原位杂交mRNA表达被广泛用于多种用途，包括研究区域组织的组织，细胞类型和脑功能活动2-5,10,12-14。以后使用，其大脑中的mRNA表达是依赖于神经活动增加，活动相关的基因通常被称为即早基因的基因。与这些用途中，我们的方法已被应用于多个物种，包括鸟类，哺乳类动物（如人类），鱼类，两栖类动物;在多个组织，包括脑，皮肤，肌肉和多个年龄段，包括幼体/新生儿，青少年，成人，并在整个胚胎部分2,3,5,15-17。我们的协议的特殊功能包括：（1）产生一个解剖特异性和特异性定量之间的平衡。量化基因表达的X射线胶片上，我们采取的数码照片的图像（如： 图1A和1B），使用“保护海港条例”toshop（;）直方图功能，在该地区的利益来衡量的像素密度和减去组织以外的胶片上的背景水平，但仍然在载玻片上2,4。为了量化在细胞水平上的表达，我们采取了图像的银粒细胞在高倍率下（40-100X， 图4）。然后，我们使用图像J的阈值和测量功能，在美国国立卫生研究院韦恩Rasband来计算图像中的银颗粒的数量，而不在载玻片上的细胞在类似的区域减去背景计数，分化的细胞数量，获得每个细胞中的表达。4,18 A值（2）它可以是相对较高的吞吐量，允许100-200幻灯片同时处理，由于紧张的盖玻片密封矿物油浴。标准的原位杂交方法需要较长的时间密封幻灯片封口膜，指甲油，和其他手段，幻灯片占用了大量的空间;（3）它是高度的敏感的低丰度的成绩单，由于硫酸葡聚糖和登哈特的杂交缓冲2,13的解决方案;（4）在暗视野成像的方法产生高对比度的图像，由于在解剖显微镜4,5暗场照明下图片。此外，它可以让敏感的微小变化，如在活动依赖的基因表达，基因表达的检测，以确定特定的大脑激活区域的19个具体行为的感知和生产过程中。相对非放射性协议的限制，后者是在蜂窝决议和细胞内的基因的位置清晰，并与乳液的工作是非常敏感的操纵和光。这是可能的，结合我们的方法与其他方法，如非放射性原位杂交标记mRNA表达多个基因在同一组织10,17,20。它可以结合免疫细胞化学标注上相同的样品RNA和蛋白表达，以合作，本地化与某些类型的细胞的基因10。这种双标记实验所需的协议修改描述中引用的参考文献。 总之，我们的做法有利于细胞的基因表达的时间和位置的认识，以便了解区域组织，组织功能活动，和基因功能。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>醋酸酐</td><td>厂商VWR </td><td> MK242002 </td><td></td></tr><tr><td>氯仿</td><td>厂商VWR </td><td> BDH1109 </td><td></td></tr><tr><td>醋酸甲酚紫</td><td>西格玛</td><td> C5042 </td><td></td></tr><tr><td>低温恒温器</td><td>热科学</td><td> MICROM HM550 </td><td></td></tr><tr><td>甲酰胺</td><td>西格玛</td><td> F9037 </td><td></td></tr><tr><td>数码地面电视</td><td> Promega公司</td><td> P1171 </td><td> 100MM </td></tr><tr><td> EDTA的</td><td>西格玛</td><td>海关</td><td></td></tr><tr><td>嵌入模具</td><td>厂商VWR </td><td> 15160-215 </td><td></td></tr><tr><td>费舍尔品牌Superfrost加幻灯片</td><td> Fisher Scientific则</td><td> 22-034-979 </TD&gt; <td></td></tr><tr><td>甲醛</td><td>厂商VWR </td><td> BDH0506-4LP </td><td></td></tr><tr><td>甲酰胺</td><td>西格玛</td><td> F7508 </td><td></td></tr><tr><td> GENECLEAN套件</td><td> Q - 生物基因</td><td> 1001-200 </td><td></td></tr><tr><td>柯达百玛士议员电影</td><td>西格玛</td><td> Z350370 </td><td></td></tr><tr><td>柯达非关税壁垒乳液</td><td> Carestream Health公司</td><td> 8895666 </td><td></td></tr><tr><td>柯达专业开发D19中</td><td>柯达</td><td> 1462593 </td><td></td></tr><tr><td>柯达专业定影液</td><td>柯达</td><td> 1971746 </td><td></td></tr><tr><td> β-巯基乙醇</td><td> Calbiochem公司</td><td> 444203 </td><td></td></tr><tr><td>矿物油</td><td>厂商VWR </td><td> IC15169491 </td><td></td></tr><tr><td>氢氧化钠</td><td>厂商VWR </td><td> SX0600-1 </td><td></td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> 76240 </td><td></td></tr><tr><td>聚一</td><td> Invitrogen公司</td><td> POLYA.GF </td><td></td></tr><tr><td> RATP </td><td> Promega公司</td><td> P1132 </td><td> 10MM </td></tr><tr><td> RCTP </td><td> Promega公司</td><td> P1142 </td><td> 10MM </td></tr><tr><td> rGTP </td><td> Promega公司</td><td> P1152 </td><td> 10MM </td></tr><tr><td> RNasin </td><td> Promega公司</td><td> N2111 </td><td> 40Units/μl </td></tr><tr><td>第35双绞线</td><td>珀金埃尔默</td><td> NEG039C001MC </td><td></td></tr><tr><td>安全解决解决方案</td><td>安全解决研究产品的国际</td><td> 111177 </td><td></td></tr><tr><td>醋酸钠</td><td>西格玛</td><td> S7899 </td><td> 3M </td></tr><tr><td>磷酸钠二元</td><td>西格玛</td><td> S3264 </td><td></td></tr><tr><td>钠磷酸二氢</td><td>西格玛</td><td> S3139 </td><td></td></tr><tr><td> SP6的RNA聚合酶</td><td> Promega公司</td><td> P1085 </td><td></td></tr><tr><td>染色的金属架</td><td>电子显微镜科学</td><td> 70312-54 </td><td></td></tr><tr><td> T7 RNA聚合酶</td><td> Promega公司</td><td> P2075 </td><td></td></tr><tr><td>组织-TEK华侨城</td><td>樱花</td><td> 4583 </td><td></td></tr><tr><td> 5X转录缓冲</td><td> Promega公司</td><td> P1181 </td><td></td></tr><tr><td>三乙醇胺</td><td>厂商VWR </td><td> IC15216391 </td><td></td></tr><tr><td>的Tris-HCl（1米，pH值8.0） </td><td>厂商VWR </td><td> 101449-446 </td><td></td></tr><tr><td>氨酰tRNA </td><td>罗氏公司</td><td> 10109509001 </td><td></td></tr></tbody></table>解决方案： <ol><li> riboprobes使试剂：1.5μLDNA模板（0.2-0.3微克/微升），2μL的5倍优化转录缓冲区，100MM的DTT 1μL（用聚合酶购自Promega公司，混合以及在室温下），RNasin（40单位/μL）0.3μL，1.5 AGC核苷酸组合解决方案，第35双绞线3.5μL，1μLRNA聚合酶液。带来高达10μL无核酸酶水。 </li><li> AGC核苷酸组合的解决方案：为10mm ATP，GTP和CTP一起混合等量。 </li><li>醋酸钠乙醇沉淀除去免费的第35条双绞线：40μL酶和DNA酶自由水，5μL3M醋酸钠，100％乙醇和125μL。 </li><li>杂交缓冲液（10毫升股票）：100％的去离子甲酰胺，600微升5M氯化钠，1M的Tris-HCl（pH值= 8.0）5毫升，240微升0.5M EDTA的（pH值= 8.0），100μl100X Denhart的解决方案的， 100μL，250μL，125μL20毫克/毫升聚一，1克硫酸葡聚糖钠20mg/mL时tRNA的1M的数码地面电视。加入DEPC处理水使总体积为10毫升。大力摇晃，然后在55°C孵育，直到所有的硫酸葡聚糖钠溶解。杂交缓冲存储-20℃，这是好的〜6个月。 </li><li>缓冲的4％多聚甲醛溶液：在760毫升蒸馏水中加入多聚甲醛指定一个烧瓶，加热到50°C的一个热点板块，而搅拌40多聚甲醛Ğ。 10N氢氧化钠加入320μL，以帮助溶解多聚甲醛。溶解后（约10分钟），加100毫升10X PBS，带音量，直到1公升蒸馏水。搅拌和加热直到溶解多聚甲醛的解决方案。 pH值应为7.4。 </li><li>乙酰缓冲区：三乙醇胺13.6毫升加2.52毫升醋酸酐在1升蒸馏水。 </li><li> 20X SSPE的解决方案：3M氯化钠，200毫米的NaH 2 PO 4-H 2 O和200毫米EDTA在蒸馏水。调整10N NaOH溶液的pH值7.4的解决方案。 </li><li> 10X PBS缓冲液：80克氯化钠，氯化钾的Na 2 HPO 414.4克2.0克，锦2 2.4G PO在蒸馏水4。调整pH值7.0的解决方案，使总体积为1精简版河</li><li>第二次洗涤液：0.1％β-巯基乙醇和2X SSPE的50％甲酰胺</li><li>甲酚紫醋酸溶液：自来水甲酚紫3％醋酸（蒸馏水防止良好的染色），溶解在瓶在室温下搅拌过夜。通过为1mm滤纸滤纸和布氏漏斗过滤真空吸。 </li></ol>

radioactive in situ hybridization for detecting diverse gene expression patterns in tissue

认识的时间，水平，细胞定位，一个基因表达的细胞类型，有助于我们了解基因的功能。这些功能都可以在原位杂交基因在细胞内完成。在这里，我们提出修改克莱顿等人 （1988）已成功地在实验室多年，特别是成年脊椎动物的大脑2-5 原位杂交方法的放射性。长互补的RNA（cRNA的）探针的靶序列，可以检测低丰度转录6,7。纳入到cRNA探针的放射性核苷酸允许进一步的检测灵敏度低丰度转录和定量分析，无论是由光敏感的X光片或乳液涂在组织。这些检测方法提供了一个长期的记录靶基因的表达。与非放射性探针冰毒消耗臭氧层物质，如地高辛标记的放射性探针杂交方法不需要使用的HRP-抗体和/或TSA试剂盒检测低丰度转录的多个扩增步骤。因此，这种方法提供了一个定量分析的信号强度和有针对性的表达量之间的线性关系。它可以同时处理100-200幻灯片。它的工作原理以及不同发育阶段的胚胎。最发育的基因表达研究使用整个胚胎和非放射性的方法，在8,9部分，是因为胚胎组织比成人组织更脆弱，减少细胞间的凝聚力，使组织切片的细胞群之间的界限很难看到。相比之下，我们的放射性的方法，由于更大范围的灵敏度，是组织地区之间的基因表达的决议能够得到更高的对比度，使其更容易看到种群之间的界限。使用这种方法，研究人员可以揭示一种新发现的基因可能的意义，并进一步预测感兴趣的基因的功能。

Watch this Scientific Journal Video about Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue at JoVE.com

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

该协议被成功地用于定量检测水平和mRNA表达的空间格局，在跨多个组织的脊椎动物物种类型。该方法可以检测低丰度的成绩单，并允许数以百计的幻灯片，同时处理。我们目前使用的禽流感为例，胚胎的大脑形成的表达分析这个协议。

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. ...

radioactive-situ-hybridization-for-detecting-diverse-gene-expression

Research

JoVE Journal

Biology

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

15.6K 次观看.  Duke University. 该协议被成功地用于定量检测水平和mRNA表达的空间格局，在跨多个组织的脊椎动物物种类型。该方法可以检测低丰度的成绩单，并允许数以百计的幻灯片，同时处理。我们目前使用的禽流感为例，胚胎的大脑形成的表达分析这个协议。

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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

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 were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

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

科研

生物学

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.
Anna Karlgren and Jenny Carlsson contributed equally to this study.
Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundstr&#246;m at Jens.Sundstrom@vbsg.slu.se

Non-radioactive in situ Hybridization Protocol Applicable for Norway Spruce and a Range of Plant Species

We describe a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including Norway spruce. With just a few adjustments, including altered RNase treatment and proteinase K concentration, the protocol may be used in studies of different tissues and species.

非放射性原位挪威云杉杂交议定书适用的植物种类范围

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of ...

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

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

高通量原位杂交方法表征在胎鼠下泌尿生殖道的基因表达模式

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

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 ...

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 ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...