We thank our colleagues for cDNA plasmids for template generation. The authors' work is supported by the Swedish Cancer Foundation (CAN 2010/553), the Swedish Foundation for International Cooperation in Research and Higher Education (IG2011-2042) and the Knut and Alice Wallenberg Foundation (KAW2012.0058).

通过体外转录1.标记RNA探针的<ol><li>装配在无RNase的条件下1.5ml的微型管在体外转录反应 :10.5微升DEPC处理过的H 2 O的，4.0微升5×转录缓冲液，1.0微升（1微克）线性化，纯化的模板DNA在DEPC处理的H 2 O的， 1.3微升NTP组合，0.7微升半抗原标记的UTP，0.5微升的RiboLock RNA酶抑制剂，2.0微升RNA聚合酶。 注意:最终反应体积为20微升。 </li><li>取决于模板的启动子序列用T7，T3或SP6 RNA聚合酶的体外转录 （见参考文献17）。 </li><li>选择地高辛-11-UTP，生物素-16-UTP，或荧光素-12-UTP作为标签的RNA探针。欲了解更多详细信息，请参阅讨论的半抗原，标签和探针浓度的讨论。 </li><li>混合转录反应和降速很快。 </li><li>让抄写3小时，在37℃。 </li><li>加入1μl无RNA酶的DNA酶I为去除模板DNA的转录反应，拌匀，孵育15分钟，在37℃。 </li><li>调整用DEPC处理过的H 2 O的样本量为200微升。 </li><li>加入100微升（0.5体积）醋酸7.5甲酸铵和600微升（3体积）乙醇沉淀处理标记的RNA探针。孵育在室温30分钟。 <ol><li>不要用冰冷的乙醇，因为这可能会导致非法人核苷酸不必要的沉淀。 </li></ol></li><li>降速中以最大速度（20800×g离心）为30分钟的温度控制离心机转录的RNA在20℃。小心吸上清液。 </li><li>在20800×g离心+20℃时洗涤所得沉淀用70％乙醇在室温和离心10分钟。 </li><li>除去上清液，小心不要意外吸出松散的颗粒。让沉淀空气干燥在翻开盖了几分钟，在室温下的微管。 </li><li>派息解决所得到的在100μlDEPC处理的H 2 O的小球</li><li>添加300微升预杂交缓冲液（50％体积/体积的去离子甲酰胺，5×SSC，50微克/毫升肝素的钠盐，0.1％体积/体积的吐温20，5毫克/毫升朊假丝核糖核酸），并存储在-20℃下。探针可以长期储存在-20℃。 注意:对于新转录的探针稀3微升半抗原标记的RNA探针在100μl杂交缓冲液的第一测试以获得所述WISH实验中的最终浓度。 </li></ol> 2.加工胚中使用插入的注意:使用微管或多孔板，用于通过不同的孵育步骤处理的胚胎承载通过交换溶液的过程中不小心吸他们失去显著量胚胎的危险。为了避免这种损失，可能会有所帮助使用筐（如Netwe​​ll插页），用于通过程序执行胚胎样品。这是聚苯乙烯serts与聚酯网在底部（图2A），在其上加工过程中的胚胎休息。 <ol><li>为WISH过程中处理的胚胎，组装在一个12孔板（图2B）的插入和加2ml适当溶液每个孔中。 </li><li>移液器胚胎成使用蓝色尖插入。确保所有的胚胎都浸在液体中。 </li><li>当孵育时间结束发生在另一个12孔板中，其中充满用于下一步骤的溶液中的含胚插入。 <ol><li>使用的载体和手柄（ 图2C），以向上从一个溶液转移到12个样品的时间到另一个（ 图2D）。 注意:为了减少所需的量，使用2.0毫升微管进行预杂交/杂交步骤（见步骤4.1）和24孔板染色反应（步骤6.2）。杂交和染色反应在100体积进行1;升和400微升，分别。 </li></ol></li></ol> 3.准备胚胎的杂交<ol><li>收集，dechorionate，修复，并用标准程序14,18 devitellinize胚胎。将制备的胚胎通常存储在甲醇中于-20℃。 </li><li>所用转印胚胎原位杂交至RT，并分配到一个12孔板填充每孔2毫升甲醇中的插入物。 </li><li>通过降低甲醇系列在室温补充水分胚胎。每次孵育胚胎3分钟在:（ⅰ）75％的甲醇中的1×PBS（ⅱ）50％甲醇中的1×PBS，0.1％Tween-20的（ⅲ）中的1×PBS的25％甲醇，0.1％吐温-20（ⅳ ）的1×PBS，0.1％吐温-20（v）的1倍PBS，0.1％吐温-20。 </li><li>预固定在4％多聚甲醛的胚胎中的1×PBS进行20分钟，在室温。 </li><li>通过4次洗涤除去固定液3分钟在1×PBS，0.1％吐温-20在RT。同时解冻并准备蛋白酶K和甘氨酸的工作方案。 </li><li&gt;在新鲜蛋白酶K透胚胎（10微克/毫升到50微克/毫升）中的1×PBS，0.1％Tween-20的3分钟在室温。 注意:蛋白酶K的治疗是胚胎固定的力量平衡。例如，如果预固定步骤（3.4）被省略，较软的蛋白酶K处理可以应用。 </li><li>停止由两个1分钟漂洗反应在2毫克/毫升甘氨酸的1×PBS，0.1％Tween-20的于RT。 </li><li>后固定在4％多聚甲醛的胚胎中的1×PBS进行20分钟，在室温。 </li><li>接着，通过洗涤除去多聚甲醛固定液四倍3分钟在1×PBS，0.1％Tween-20的于RT。 </li><li>转印后固定胚胎预杂交缓冲液。商店的胚胎在预杂交缓冲液中在-20℃或直接进行探针的杂交。 </li></ol> 4.杂交探针的<ol><li>储存在-20℃下在预杂交缓冲液至RT转移胚胎。分布在预杂交缓冲液中的胚胎成2.0毫升microtub上课。 </li><li> 30分钟至少，在预杂交缓冲液预杂交胚胎的样品在65℃。 注意:对于预杂交和杂交步骤水浴被使用。在预杂交有时间通过​​合成多达3的反义RNA探针，每个特异于不同基因转录物，并标有一个不同的半抗原来制备探针混合物。 </li><li>以获得探针混合物，加在一起适当量（通常在1微升至6微升）每个期望的原始半抗原标记的反义RNA在100μl杂交缓冲液（50％体积/体积的去离子甲酰胺，5×SSC，50微克/毫升探针肝素钠盐，0.1％体积/体积的吐温20，5毫克/毫升朊假丝核糖核酸，5％重量/体积的葡聚糖硫酸盐）。 注:加入硫酸葡聚糖是可选的。 </li><li>变性反义RNA探针混合物在加热块在80℃下进行5分钟。随后直接传送变性探针混合物至65℃。 </li><li>除去大多数预杂交的从胚胎样本缓冲区，并添加变性探针混合物。 </li><li>让胚胎样本杂交探针混合物O / N在65℃。 注意:温度在55℃至65℃不等常规用于杂交，前，后的杂交步骤。杂交在65℃下提供最严格的条件，并使用较低的温度时，建议的情况下的背景问题。如果没有背景的问题，在55℃杂交，建议，它提供了增强的信号强度和改善的胚胎完整性的优点。 </li><li>第二天早上，第一严紧洗涤溶液转移至65℃，以确保它们被加热到正确的温度的时间。 </li><li>转移杂交探针混合物与来自2.0毫升微管胚胎成12孔板含2ml预热杂交洗涤缓冲液的插入物（在2×SSC 50％甲酰胺，0.1％吐温-20）。把这个包含的EM的12孔板在烘箱bryo样品孵育20分钟，在65℃。 </li><li>移植胚胎的样品至2ml新的杂交洗涤缓冲液孵育20分钟，在65℃。再一次重复此步骤。 </li><li>通过20分钟的洗涤，在65℃在2×SSC中取出多余的探针，一次，0.1％吐温20和0.2×SSC的三倍，0.1％吐温-20。 </li><li>转移胚胎的样品至1x PBS，0.1％吐温-20在RT。 </li><li>孵育在封闭缓冲液为至少在室温30分钟（8％绵羊中的1×PBS，0.1％Tween-20的血清）的胚胎。 </li></ol> 5.抗体检测半抗原标记探针注意:相对于所有的三个不同标记的探针的杂交同时，各探针被检测到相继在单独的轮抗体孵育和染色。因此，在每个检测回合只有一种抗体被使用（或者抗洋地黄毒苷AP或抗荧光素-AP或抗生物素-AP）。 <ol><li> STEP之间进行选择5.1.1至5.1​​.3和继续根据半抗原 - 标签，该标签是被检测准备适当的抗体稀释液。 注意:对于顺序检测不同的标记探针，另一种抗半抗原抗体，在每个检测周期施加。 <ol><li>用于检测的荧光素标记的探针的稀释抗荧光素-AP偶联物1:4000在封闭缓冲液。 </li><li>用于检测的地高辛标记的探针的稀释抗洋地黄毒苷-AP偶联物1:4000在封闭缓冲液。 </li><li>用于检测的生物素标记的探针的制备在一个1的抗生物素的AP缀合物溶液:4000稀释在封闭缓冲液。 </li></ol></li><li>孵育在3至4小时，在RT下选择的抗半抗原的AP溶液胚胎。另外，孵化O / N在4℃。不要同时应用所有三种抗体。 </li><li>为了除去未结合的抗体洗胚用1×PBS，0.1％Tween-20的4次，每次15分钟，在RT。 </li><li>要改变缓冲系统是h两次或次15在RT（分钟）中的TNT（0.1M的Tris-HCl pH 8.0的0.1 M氯化钠，0.1％吐温-20）。 </li></ol> 6.碱性磷酸酶显色染色<ol><li>步骤6.1.1之间6.1.4之后抗体检测选择并继续进行碱性磷酸酶染色显色反应，以获得所需的颜色信号的一个（ 见表 1）。每轮检测的不同生色底物的反应被施加到突出另一种颜色的每个转录物图案。 <ol><li>快速红染色<ol><li>对于在TT8.2 15分钟（0.1M的Tris-HCl pH值8.2，0.1％吐温-20）洗涤胚胎两次。 </li><li>虽然洗的胚胎，准备从固红TR / NAMP碱性磷酸酶底物片设置快速红染色液。 </li><li>溶解的药片在1毫升蒸馏水中设置一个缓冲片剂和涡旋获得的0.1M的Tris-HCl pH值8.2混合。 </li><li>删除快速红色TR / NAMP平板电脑进入准备牛逼里斯缓冲液和涡旋以获得固红染色溶液溶解。 </li><li>明确坚牢红染色从非溶解颗粒通过0.2μm注射器过滤溶液。 </li></ol></li><li>耐晒蓝染色<ol><li>在新鲜SB8.2染色缓冲器15分钟洗胚胎两次（0.1M的Tris-HCl pH值8.2，0.1M氯化钠，0.05M的氯化镁 ，0.1％吐温-20）。 </li><li>而洗涤胚胎，从50毫克/毫升固蓝BB（在DMF中）和50毫克/毫升磷酸盐萘酚（在DMSO中）的储备溶液制备固蓝染色溶液。 </li><li>新鲜制备0.5毫克/毫升SB8.2一个固蓝BB溶液。 </li><li>新鲜制备0.5毫克/毫升SB8.2单独萘酚磷酸盐溶液。 </li><li>滴加加入准备好的萘酚磷酸盐溶液到在不断混合下将固蓝BB溶液用涡旋。所得的最终工作浓度为0.25毫克/毫升为基底部件。 注:不同的耐晒蓝/萘酚phosp恨组合将导致不同颜色深浅（ 见表 1）。 </li></ol></li><li> BCIP / NBT染色<ol><li>在新鲜SB9.5染色缓冲器15分钟洗胚胎两次（0.1M的Tris-HCl pH为9.5，0.1M氯化钠，0.05M的氯化镁 ，0.1％吐温-20）。 </li><li>对于purpleblue色信号，刚准备BCIP / NBT染色溶液中加入1微升左旋咪唑，3.5微升BCIP每毫升SB9.5染色缓冲液4.5微升NBT拌匀。 </li></ol></li><li> INT染色<ol><li>在新鲜SB9.5染色缓冲器15分钟洗胚胎两次。 </li><li>对于yellowbrown色信号，刚准备INT染色溶液中加入1微升左旋咪唑，5微升品红，磷酸和每毫升SB9.5染色缓冲液5微升INT拌匀。 注:另外使用7.5微升/毫升BCIP / INT准备使用的染色溶液。 </li></ol></li></ol></li><li>对于染色反应移植胚胎样本到一个24孔板中。孵育于400μl在黑暗中所选择的染色液的每个样本。 </li><li>监控偶尔在立体显微镜下信号的发展。停止染色反应时所需要的信号强度获得并显著背景信号后才能看到。 </li><li>以停止显色反应，染色的胚胎转移到一个12孔平板每孔含有2 ml的TNT炸药插入。冲洗染色胚胎两次TNT。 </li><li> 10分钟在1×PBS中洗胚胎两次，0.1％吐温-20在RT以去除残余衬底。 </li></ol> 7.抗体去除/碱性磷酸酶灭活<ol><li>除去抗体和灭活碱性磷酸酶温育在2ml低pH终止溶液（0.1M甘氨酸-HCl pH值2.2，0.1％吐温-20）在室温10分钟。 </li><li> 1分钟漂洗用2ml 1×PBS，0.1％吐温-20在RT。 </li><li> 3分钟在1×PBS洗涤3次，0.1％吐温-20在RT。 </li></ol> 8.第二检测轮<ol><li>为了检测第二成绩单的分布格局进行抗体检测使用目标针对的是第二个半抗原标记抗体-AP偶联半抗原标记探针（STEP 5.1至5.4）的。 </li><li>执行碱性磷酸酶染色显色（步骤6.1至6.5）将产生一个对比色，以在第一检测轮中使用的（对于可用的颜色见表 1）的基板组合。 </li><li>在步骤7.1说明7.3卸下第二抗体-AP结合物。 </li></ol> 9.第三检测轮<ol><li>为了检测一个第三转录物的分布模式，继续使用抗体-AP偶联物特异于第三半抗原 - 标记抗体检测半抗原标记的探针（步骤5​​.1至5.4）。 </li><li>执行碱性磷酸酶染色显色（STEP 6.1至6.5）将基材组合，这不是AP合股在之前的检测回合。选择产生颜色沉淀轻松从那些先前两个检测两轮区分显色反应（用于彩色选择见表 1）。 </li></ol> 10.安装和成像<ol><li>妥善转移胚胎染色至30％甘油的PBS和孵化，直到胚胎中的甘油溶液充分浸泡。他们很平衡，当他们沉到水底。 </li><li>转移胚胎到H 2 O的70％甘油，让平衡在室温。染色的胚胎可以在4℃储存在70％甘油中。 </li><li>用于安装，吸管染色在70％甘油液滴胚胎到物体上滑动和不接触粘在滑动（图3）的隔离物。应用盖玻片。 </li><li>视图安装在解剖显微镜下的胚胎。移动所施加的盖玻片，以使胚胎旋转到所需的取向。 </li><li>注意正确定向胚胎复式显微镜与微分干涉对比光学高分辨率下（20X，40X的目标）。 </li><li>捕捉图像的数字彩色照相机和保存数字存储设备。 </li></ol>

<ol>
	<li>Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Situ Hybridization Methods. , (2015).</li><li>Kosman, D., 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=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305 (5685), 846 (2004).</li><li>Itzkovitz, S., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validating+transcripts+with+probes+and+imaging+technology.">Validating transcripts with probes and imaging technology.</a> Nature Methods. 8 (4), S12-S19 (2011).</li><li>Kwon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+fluorescence+in+situ+hybridization:+Quantitative+imaging+of+single+RNA+molecules.">Single-molecule fluorescence in situ hybridization: Quantitative imaging of single RNA molecules.</a> Bmb Reports. 46 (2), 65-72 (2013).</li><li>Speel, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robert+Feulgen+Prize+Lecture+1999.+Detection+and+amplification+systems+for+sensitive,+multiple-target+DNA+and+RNA+in+situ+hybridization:+looking+inside+cells+with+a+spectrum+of+colors.">Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors.</a> Histochem Cell Biol. 112 (2), 89-113 (1999).</li><li>Hafen, E., Levine, M., Garber, R. L., Gehring, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+in+situ+hybridization+method+for+the+detection+of+cellular+RNAs+in+Drosophila+tissue+sections+and+its+application+for+localizing+transcripts+of+the+homeotic+Antennapedia+gene+complex.">An improved in situ hybridization method for the detection of cellular RNAs in Drosophila tissue sections and its application for localizing transcripts of the homeotic Antennapedia gene complex.</a> EMBO J. 2 (4), 617-623 (1983).</li><li>Tautz, D., Pfeifle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-radioactive+in+situ+hybridization+method+for+the+localization+of+specific+RNAs+in+Drosophila+embryos+reveals+translational+control+of+the+segmentation+gene+hunchback.">A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback.</a> Chromosoma. 98 (2), 81-85 (1989).</li><li>Hartmann, C., J&#228;ckle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+relationships+between+a+novel+Drosophila+stripe+expressing+gene+and+known+segmentation+genes+by+simultaneous+visualization+of+transcript+patterns.">Spatiotemporal relationships between a novel Drosophila stripe expressing gene and known segmentation genes by simultaneous visualization of transcript patterns.</a> Chromosoma. 104 (2), 84-91 (1995).</li><li>Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+detection+of+mRNA+transcript+localizations+in+fish+and+fly+embryos+using+alkaline+phosphatase+and+beta-galactosidase+conjugated+antibodies.">Two-color detection of mRNA transcript localizations in fish and fly embryos using alkaline phosphatase and beta-galactosidase conjugated antibodies.</a> Dev Genes Evol. 209 (5), 317-321 (1999).</li><li>Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-,+two-,+and+three-color+whole-mount+in+situ+hybridization+to+Drosophila+embryos.">One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos.</a> Methods. 23 (4), 359-372 (2001).</li><li>Hauptmann, G., Gerster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+whole-mount+in+situ+hybridization+to+vertebrate+and+Drosophila+embryos.">Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos.</a> Trends Genet. 10 (8), 266 (1994).</li><li>Hauptmann, G., Gerster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolour+whole-mount+in+situ+hybridization+to+Drosophila+embryos.">Multicolour whole-mount in situ hybridization to Drosophila embryos.</a> Development Genes and Evolution. 206 (4), 292-295 (1996).</li><li>O'Neill, J. W., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-label+in+situ+hybridization+using+biotin+and+digoxigenin-tagged+RNA+probes.">Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes.</a> Biotechniques. 17 (5), 870-875 (1994).</li><li>S&#246;ll, I., Hauptmann, G., G, H. a. u. p. t. m. a. n. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolored+visualization+of+transcript+distributions+in+Drosophila+embryos.">Multicolored visualization of transcript distributions in Drosophila embryos.</a> Neuromethods. 99, In situ hybridization methods, 45-59 (2015).</li><li>Bobrow, M. N., Harris, T. D., Shaughnessy, K. J., Litt, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalyzed+reporter+deposition,+a+novel+method+of+signal+amplification.+Application+to+immunoassays.">Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays.</a> J Immunol Methods. 125 (1-2), 279-285 (1989).</li><li>Hauptmann, G., Gerster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+whole-mount+in+situ+hybridization.">Multicolor whole-mount in situ hybridization.</a> Methods Mol Biol. 137, 139-148 (2000).</li><li>S&#246;ll, I., Hauptmann, G., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+and+automated+whole-mount+in+situ+hybridization+for+systematic+gene+expression+analysis+in+embryonic+zebrafish+forebrain.">Manual and automated whole-mount in situ hybridization for systematic gene expression analysis in embryonic zebrafish forebrain.</a> Neuromethods. 99, In situ hybridization methods, 171-206 (2015).</li><li>Bergalet, J., et al., Hauptmann, G., 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=Subcellular+transcript+localization+in+Drosophila+embryos+and+tissues+visualized+by+multiplex+FISH.">Subcellular transcript localization in Drosophila embryos and tissues visualized by multiplex FISH.</a> Neuromethods. 99, In situ hybridization methods, 369-391 (2015).</li><li>Walldorf, U., Gehring, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empty+spiracles,+a+gap+gene+containing+a+homeobox+involved+in+Drosophila+head+development.">Empty spiracles, a gap gene containing a homeobox involved in Drosophila head development.</a> Embo J. 11 (6), 2247-2259 (1992).</li><li>Hafen, E., Kuroiwa, A., Gehring, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+distribution+of+transcripts+from+the+segmentation+gene+fushi+tarazu+during+Drosophila+embryonic+development.">Spatial distribution of transcripts from the segmentation gene fushi tarazu during Drosophila embryonic development.</a> Cell. 37 (3), 833-841 (1984).</li><li>Grossniklaus, U., Pearson, R. K., Gehring, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+sloppy+paired+locus+encodes+two+proteins+involved+in+segmentation+that+show+homology+to+mammalian+transcription+factors.">The Drosophila sloppy paired locus encodes two proteins involved in segmentation that show homology to mammalian transcription factors.</a> Genes Dev. 6 (6), 1030-1051 (1992).</li><li>Hauptmann, G., Gerster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+gene+expression+patterns+reveal+transverse+and+longitudinal+subdivisions+of+the+embryonic+zebrafish+forebrain.">Regulatory gene expression patterns reveal transverse and longitudinal subdivisions of the embryonic zebrafish forebrain.</a> Mech Dev. 91 (1-2), 108-118 (2000).</li><li>Long, S., Rebagliati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+two-color+whole-mount+in+situ+hybridizations+using+digoxygenin-+and+dinitrophenol-labeled+RNA+probes.">Sensitive two-color whole-mount in situ hybridizations using digoxygenin- and dinitrophenol-labeled RNA probes.</a> Biotechniques. 32 (3), 494-500 (2002).</li><li>Lauter, G., S&#246;ll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+fluorescent+in+situ+hybridization+to+define+abutting+and+overlapping+gene+expression+in+the+embryonic+zebrafish+brain.">Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain.</a> Neural Dev. 6 (1), 10 (2011).</li><li>Lauter, G., S&#246;ll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+whole-mount+fluorescent+in+situ+hybridization+in+zebrafish+using+enhanced+tyramide+signal+amplification.">Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification.</a> Methods Mol Biol. 1082, 175-185 (2014).</li><li>Chen, J., Laramore, C., Shifman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triple-labeling+whole-mount+in+situ+hybridization+method+for+analysis+of+overlapping+gene+expression+in+brain+tissue+with+high+level+of+autofluorescence.">Triple-labeling whole-mount in situ hybridization method for analysis of overlapping gene expression in brain tissue with high level of autofluorescence.</a> J Cytol Histol. , S3-S011 (2015).</li><li>Murdoch, A., Jenkinson, E. J., Johnson, G. D., Owen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alkaline+phosphatase-fast+red,+a+new+fluorescent+label.+Application+in+double+labelling+for+cell+cycle+analysis.">Alkaline phosphatase-fast red, a new fluorescent label. Application in double labelling for cell cycle analysis.</a> J Immunol Methods. 132 (1), 45-49 (1990).</li><li>Hauptmann, G., Lauter, G., S&#246;ll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+alkaline+phosphatase-mediated+azo+dye+staining+for+dual+fluorescent+in+situ+hybridization+in+zebrafish.">Application of alkaline phosphatase-mediated azo dye staining for dual fluorescent in situ hybridization in zebrafish.</a> Neuromethods. 99, In situ hybridization methods, 393-404 (2015).</li><li>Lauter, G., S&#246;ll, I., Hauptmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-color+fluorescent+in+situ+hybridization+in+the+embryonic+zebrafish+brain+using+differential+detection+systems.">Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.</a> BMC Dev Biol. 11, 43 (2011).</li></ol>

The authors have nothing to disclose.

原位杂交（ISH）与放射性标记的核酸探针通常用于检测RNA的定位上的组织切片。放射性原位杂交的方法，但是很费时间，较不敏感，并且不允许升值的全坐骑完整成绩单分布格局。与此相反，本文中所描述的MC-WISH方法允许多基因表达的结构域在完整的胚胎内的对比色的直接比较。 MC-WISH具有的优点在于组织学方面是立即显而易见的，简单地通过标准明显微镜观察。这提供了可能性地形之间的相互关系以高精度基因表达域和限定在一个快速和可靠的方式独特和重叠的表达位点。另一方面，复用荧光原位杂交（FISH）是更强大的相对于敏感性和分辨率，并优选使用，如果蜂窝或者甚至亚细胞再检测mRNA的溶液是必需的。 转录物的广谱已映射由MC-WISH表明几乎所有转录物，不仅可以在胚胎而且在幼虫和成人组织和物种比果蝇其他被分析。事实上，所描述的过程的详细这里果蝇 ，工作方式类似于高效与斑马鱼胚胎11,16,22。此外，在MC-WISH方法可适应多种无脊椎动物和脊椎动物的胚胎和组织标本的。 半抗原标签和探针浓度我们经常使用的荧光素，地高辛和生物素的RNA探针的半抗原标签。另外，二硝基酚标记的探针可以应用，已在斑马鱼WISH实验23-25 ​​介绍。荧光素标记的探针显示相较于其它半抗原标记在较小的敏感性，从而使荧光素，最好使用˚F或检测丰富的成绩单。每个新转录探针在一个单一的WISH实验，其中其性能进行了评价或者使用BCIP / NBT或固红作为底物首先测试。探针浓度被认为是最佳的时候没有背景发展提供强有力的信号，在分钟内实现到几个小时。 以确保感兴趣的表达结构域已经在其通过的MC-WISH充分的程度被检测到，它是必不可少的可视化通过使用最敏感的基底结合，BCIP / NBT单标签WISH实验每个转录物的图案。这确保了弱表达结构域在多靶实验不被忽视。为了补偿其他底物组合的灵敏度更低它比标准BCIP / NBT染色必须使用加倍（至三倍）探针浓度。 低pH值失活在低pH灭活步骤可导致从而减少了在第二和第三轮染色信号检测反义半抗原标记的RNA探针/义mRNA的杂交部分崩解。为了最大限度地减少在灵敏度损失，失活的步骤是尽可能短。我们经历了一个10分钟的培养时间在低pH值足以消除AP-活动。随后迅速洗涤​​大批量确保迅速稀释的未结合的抗体-AP偶联物和防止再结合到半抗原探针。替代方法包括多聚甲醛固定和热失活。然而，一些颜色析出物不热稳定性和多聚甲醛固定可能不足以对AP的完全失活。第一应用抗体-AP偶联物的不完全的失活可导致重新可视化在下列检测轮导致假阳性重叠在表达与第二mRNA种类待检测的mRNA的图案。因此，在一个对照实验中第Ë胚分成两部分的第一应用抗体-AP偶联物的失活后。第二抗体 - 缀合物的AP从控制级分省略，并且不应当产生在第二颜色反应的信号。然而，如果第二颜色反应产生相应于第一个的信号的分布图案，则​​灭活过程是没有效率。在这种情况下，在低pH值终止溶液的温育时间应延长为下一个实验。 AP基板应用订单由于灵敏度下降以后每次轮检测的，最好是之前更丰富的转录检测低丰度的mRNA。此外，固红和固蓝底物组合是显著比purpleblue BCIP / NBT染色不太敏感，从而使快速染料在第一染色轮优选适用于检测强表达转录物。这也可为h作为后续的BCIP / NBT染色可以监测和停止时间的purpleblue信号变得太暗相对于打火机快速染料之前的优点。因此，在一个标准的两色实验，第一强表达的mRNA是通过固红检测和第二较弱的一个由BCIP / NBT。作为替代快速染料的黄色INT底物组合可以应用，然而其产生沉淀物，经过一段时间变得扩散。因此BCIP / INT专门应用在最后一轮染色。因此在三色实验中，我们经常使用的第一个快速红色，第二BCIP / NBT（或快速蓝色）和第三的INT基板结合。注意，最好是施加的INT时尽快拍摄染色的胚胎越好。 荧光检测偶氮染料它可以是有时难以识别重叠的表达模式，当一个较深的颜色沉淀阴影打火机之一。 F或者例如，强烈发展的BCIP / NBT沉淀会掩盖较轻的快速染料的信号。来解决这个问题的一个方法是将每个染色后立即捕获图像和接下来的检测回合之前除去所施加的颜色的沉淀物。在这种情况下，醇可溶偶氮染料（快速红）和INT沉淀用乙醇洗涤除去所述第一和第二检测回合后 ​​，分别和BCIP / NBT染色作为最后的AP- 基片26施加。另一种可能性是利用偶氮染料的荧光特性的优势。耐晒大红可以用罗丹明过滤器进行可视化设置27，同时耐晒蓝可以与远红外滤光片28,29可以观察到。比较或生色和荧光图像的覆盖可以揭示共distribution.Using这种方法，BCIP / NBT信号发展的网站已在时间要被停止，以便它不会变得过于致密，淬灭偶氮染料的荧光信号反应产物。

原位杂交（ISH）是用于检测和RNA转录的定位在形态学方面，内细胞，组织和生物体1的标准方法。通过原位杂交程序产生的信号通过放射性​​，荧光和发色检测系统通常可视化。近年来在荧光ISH显著技术进步（FISH）2导致显着提高的灵敏度和分辨率，从而允许检测和RNA表达的定量在单细胞和亚细胞水平和RNA可视至多单个分子3,4。而复杂的单分子的FISH方法用于更专门的应用，显色原位杂交是普遍如在研究和临床诊断原位检测方法的常规的RNA。对于发色检测酶沉淀反应时，能产生明显的产品在网站对比色杂交5。这具有的RNA的可视化可以通过常规的组织学染色和形态​​学方面进行组合的优点是立即通过标准明显微镜明显。此外，许多应用的彩色基片产生的沉淀物，其是稳定的有机和/或含水安装介质，以便永久样品制剂可以得到5。 在果蝇中，初始ISH协议应用于放射性同位素标记探针检测转录的切片材料6。虽然难以从组织切片以重建整个胚或器官系统的完整转录图案，显色原位杂交程序与非放射性标记的探针的应用使得有可能全局检测核糖核酸分布在全坐骑7。虽然显色原位杂交的许多变型存在，在典型原位杂交（WISH）协议HYB整个安装ridized半抗原标记的探针通过缀合至一个报告酶和mRNA转录由沉淀色原可视抗半抗原抗体检测。 由记者产生不同颜色的沉淀物调色板酶碱性磷酸酶（AP），辣根过氧化物酶（POD），和β-半乳糖苷酶（GAL）允许在一个独特的检测的多个目标和相同的样品8-14。然而，过氧化物酶的活性仅持续有限的时间内和在GAL比色反应有点不太敏感，因此无需额外的（酪酰胺）信号放大15的低丰度转录物的检测可以是用这些酶有挑战性。与此相反，AP的持久活性允许长效基底周转和高信噪比。因此，使用具有不同颜色的底物的AP报告酶序列检测已经证明是成功的，在有效和在单胚胎10-12,16鲜明检测高达三种不同的转录。 对于这种多目标生色WISH（MC-WISH）方法（图1）14，通过体外转录产生并标有可用的半抗原的其中一个标签标记的反义RNA探针。胚胎是甲醛固定和甲醇处理和蛋白酶K消化透。胚胎的杂交同时进行多达三个不同地标记的反义核糖核酸探针的每种特异于不同的基因。除去未结合的探针通过严格洗涤后每半抗原标签可视化在一个单独的一轮检测。单个检测轮由胚胎与抗半抗原抗体耦合到AP和核糖核酸可视化通过施加的AP-基板产生的局部稳定的颜色的沉淀物的温育。抗体检测和染色，所施加的抗体-AP conju后门由低pH值洗涤除去。在多色实验，每轮检测采用靶向不同的半抗原-标记的抗体，每个转录模式由不同的颜色基板（表1）可视化。胚胎被安装在甘油和使用微分干涉对比（DIC）光学高分辨率化合物显微镜下成像。

<table><tbody><tr><td>Deionized, diethylpyrocarbonate (DEPC) treated water</td><td>Thermo Scientific</td><td>R0603</td><td></td></tr><tr><td>T7 RNA Polymerase&amp;nbsp;</td><td>Thermo Scientific</td><td>EP0111</td><td></td></tr><tr><td>T3 RNA Polymerase&amp;nbsp;</td><td>Thermo Scientific</td><td>EP0101</td><td></td></tr><tr><td>SP6 RNA Polymerase&amp;nbsp;</td><td>Thermo Scientific</td><td>EP0131</td><td></td></tr><tr><td>RiboLock RNase Inhibitor&amp;nbsp;</td><td>Thermo Scientific</td><td>EO0381</td><td></td></tr><tr><td>DNase I, RNase-free&amp;nbsp;</td><td>Thermo Scientific</td><td>EN0521</td><td></td></tr><tr><td>NTP Set</td><td>Thermo Scientific</td><td>R0481</td><td></td></tr><tr><td>Digoxigenin-11-UTP</td><td>Roche</td><td>11209256910</td><td></td></tr><tr><td>Fluorescein-12-UTP</td><td>Roche</td><td>11427857910</td><td></td></tr><tr><td>Biotin-16-UTP</td><td>Roche</td><td>11388908910</td><td></td></tr><tr><td>Ammonium acetate</td><td>Applichem</td><td>A2936</td><td></td></tr><tr><td>Ethanol, absolute&amp;nbsp;</td><td>Merck</td><td>100983</td><td></td></tr><tr><td>di-Sodium hydrogen phosphate</td><td>Scharlau</td><td>SO0339</td><td></td></tr><tr><td>Sodium dihydrogen phosphate</td><td>Scharlau</td><td>SO0331</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>32213</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Scientific</td><td>EO0491</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7126</td><td></td></tr><tr><td>Hydrochloric acid&amp;nbsp;</td><td>Merck</td><td>100317</td><td></td></tr><tr><td>tri-Sodium citrate</td><td>Scharlau</td><td>SO0200</td><td></td></tr><tr><td>deionized formamide</td><td>Applichem</td><td>A2156</td><td></td></tr><tr><td>RNA type VI from torula yeast</td><td>Sigma</td><td>R6625</td><td></td></tr><tr><td>Heparin sodium salt</td><td>Applichem</td><td>A3004</td><td></td></tr><tr><td>Dextran sulfate sodium salt</td><td>Sigma</td><td>D6001</td><td></td></tr><tr><td>Sheep serum</td><td>Sigma</td><td>S2263</td><td></td></tr><tr><td>Sheep anti-biotin-AP Fab fragments&amp;nbsp;</td><td>Roche</td><td>11426303001</td><td></td></tr><tr><td>Sheep anti-digoxigenin-alkaline phosphatase (AP) Fab fragments</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>Sheep anti-fluorescein-AP Fab fragments</td><td>Roche</td><td>11426303001</td><td></td></tr><tr><td>Rabbit anti-dinitrophenyl-AP antibody</td><td>Vector laboratories</td><td>MB-3100</td><td></td></tr><tr><td>Tris-(hydroxymethyl)-aminomethane</td><td>Scharlau</td><td>TR04241000</td><td></td></tr><tr><td>Magnesium chloride</td><td>Scharlau</td><td>MA0036</td><td></td></tr><tr><td>Sodium chloride</td><td>Scharlau</td><td>SO0227</td><td></td></tr><tr><td>Levamisole</td><td>Sigma</td><td>L9756</td><td></td></tr><tr><td>N,N-Dimethylformamide</td><td>Sigma</td><td>D4551</td><td></td></tr><tr><td>Dimethylsulfoxide</td><td>Applichem</td><td>A3006</td><td></td></tr><tr><td>Fast Red tablet set</td><td>Sigma</td><td>F4648</td><td></td></tr><tr><td>Fast Blue BB salt</td><td>Sigma</td><td>F3378</td><td></td></tr><tr><td>Naphthol-AS-MX-phosphate</td><td>Sigma</td><td>N5000</td><td></td></tr><tr><td>Naphthol-AS-GR-phosphate</td><td>Sigma</td><td>N3625</td><td></td></tr><tr><td>Naphthol-AS-BI-phosphate</td><td>Sigma</td><td>N2250</td><td></td></tr><tr><td>Magenta-Phos</td><td>Biosynth</td><td>B7550</td><td></td></tr><tr><td>INT</td><td>Sigma</td><td>I8377</td><td></td></tr><tr><td>NBT</td><td>Applichem</td><td>A1243</td><td></td></tr><tr><td>BCIP</td><td>Applichem</td><td>A1117</td><td></td></tr><tr><td>INT/BCIP solution</td><td>Roche</td><td>11681460001</td><td></td></tr><tr><td>Glycerol 86-88%</td><td>Scharlau</td><td>GL0023</td><td></td></tr><tr><td>Universal incubator</td><td>Memmert</td><td>BE400</td><td></td></tr><tr><td>Waterbath</td><td>Memmert</td><td>WB14</td><td></td></tr><tr><td>Heat block</td><td>Grant</td><td>QBT2</td><td></td></tr><tr><td>Netwell&amp;nbsp; inserts 15 mm</td><td>Corning</td><td>3477</td><td></td></tr><tr><td>Netwell carrier kit 15 mm</td><td>Corning</td><td>3520</td><td></td></tr><tr><td>12-well cell culture plate</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>24-well plate</td><td>Sarstedt</td><td>83.3922</td><td></td></tr><tr><td>1.5 ml microtube</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>2.0 ml microtube</td><td>Sarstedt</td><td>72.691</td><td></td></tr></tbody></table>

multi-target chromogenic whole-mount in situ hybridization for comparing gene expression domains in drosophila embryos

分析胚胎发育和器官，必须精确地定义如何在不同的基因的表达在空间关系彼此在期间原位活性基因调控网络。多目标生色整个安装原位杂交（MC-WISH）大大方便的基因表达模式的瞬间相比，因为它允许不同的mRNA种类的独特的可视化中相同的样品试样的对比色。这提供了可能性地形涉及基因表达的结构域彼此以高精度和界定独特的和重叠的表达位点。在所提出的协议中，我们描述了比较不同的基因表达模式在果蝇胚胎中一个MC-WISH程序。多达三个的RNA探针，每个特定的另一种基因和标记的由不同的半抗原，被同时杂交的胚胎的样品，随后通过碱检测NE磷酸酶为基础的比色免疫组化。所描述的过程，这里果蝇详细，但同样适用与斑马鱼的胚胎。

所描述的协议允许在不同颜色的多个转录模式的同时可视化。 MC-WISH提供的优点直接比较在相同的胚胎不同基因的表达模式。作为一个例子， 空气孔的表达模式（EMS）19， 富士tarazu（FTZ）20和草率配对 1（SLP1）21被直接比较果蝇胚期胚胎和可视化中的各种颜色深浅（图4） 。 不同的AP底物组合可用于获得对比色沉淀物（表1）的面板。通过快速红色和由BCIP / NBT产生的紫色沉淀产生的红色沉淀物可容易地区别，因此使用最常见LY在双色实验（ 图4A）。然而，BCIP / NBT染色可以迅速变成一个比较暗的颜色，从而使快速红色是伪装的情况下，成绩单的部分共同配送。因此，固蓝的应用程序可以是有利的，其产生蓝色，绿色和紫色的产品与NAMP，NAGP和NABP，分别为（ 表1）组合。耐晒蓝与NAMP的结合为一个淡蓝色的颜色，是从相邻或重叠的表达域快速红色产品容易辨别如下所示的环境管理体系 ，并分别显现红色和蓝色，SLP1表达位点（ 图4B）。的坚牢蓝NAGP组合提供了一个类似的强烈对比的是固红染色，以使绿色的EMS域显然来自红色SLP1表达位点（图4C）独特的。但是，如果EMS和 SLP1表达部位进行解由固蓝NABP和直接红tected，分别将所得紫色和红色沉淀物较少可辨别（图4D）。如上所述，固红和固蓝NAGP很容易区分作为在红色和绿色分别（图4D）示出由SLP1和FTZ表达。 与INT组合MagentaPhos产生黄色沉淀物，其用于揭示FTZ的节段性表达（图4C 中的E）。将黄色有色沉淀产生良好的对比度，以作为例证SLP1和FTZ与固蓝NAMP和MAG / INT分别（图4E）的检测的蓝色底物。但是，黄色沉淀，从快速红色产物少，易辨。 EMS的黄染表达域可以是很难分销商<html>从SLP1的喙表达红色（ 图 4F）所示nguished。因此，色底物的结合，必须仔细地选择为每个实验。 <img alt="图1" src="/files/ftp_upload/53830/53830fig1.jpg" /> 图1.流程图MC-的愿望。对于三个独特的成绩单图案五彩的可视化，不同的抗体结合物AP和颜色的底物组合中的每个检测周期之间进行选择。缩写:AP，碱性磷酸酶;生物，生物素;地高辛，洋地黄毒苷;萤光灯，荧光。对于其他缩写见传说图 4 和表1。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53830/53830fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> S / ftp_upload / 53830 / 53830fig2.jpg"/&gt; 图2.插入作为胚胎载体。（A）中安装在用15毫米直径和74微米的筛目大小的聚酯膜的底部插入作为果蝇胚胎的载体。 （B）中的插入件被组装在12孔板中，其中作为水库托盘的各种解决方案的功能。 （ 三）提供运营商和把手允许的（D）处理12插入/胚胎样本同时进行。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53830/53830fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53830/53830fig3.jpg" /> 图3.安装染色胚胎。胚胎被安装在70％甘油的两叠盖玻片，这是间隔功能的下降。该应用程序的温和运动谎称大盖玻片有助于旋转安装的胚胎成观测和摄影所需方向。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53830/53830fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53830/53830fig4.jpg" /> 图4 mRNA转录在果蝇胚胎 的 彩色可视化。的MC-WISH实验果蝇胚胎例子显示，从横向的看法。 空气孔的表达方式（EMS）， 富士tarazu（FTZ）和马虎配对1（SLP 1）由不同的AP显色底的组合，这是显示在每个面板上的可视化。由（A） 保税区萤光灯和 EMS DIG（检出成绩单<stroNG&gt; B）SLP1萤光灯，EMS BIO和自由贸易区掏，（C）EMS BIO，SLP1萤光灯和自由贸易区掏，（D） 保税区生物，SLP1萤光灯和 EMS DIG，（E）SLP1萤光灯，EMS DIG和保税区生物，（F） 保税区生物，SLP1萤光灯和 EMS DIG标记探针。半抗原标记的探针进行免疫组化检测陆续在上市订单。简称:FB耐晒蓝; FR，快速红色药片。对于其他缩写见传说图 1 和表1。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53830/53830fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 基材组合 </td><td> 添加到1ml的AP缓冲 </td><td> Concentra化[微克/毫升] </td><td> AP缓冲 </td><td> 颜色 </td><td> 信号灵敏度 </td></tr><tr><td> BCIP NBT </td><td> 3.5微升4.5微升</td><td> 175 337.5 </td><td> SB9.5 </td><td>紫色</td><td> +++ </td></tr><tr><td> MAG INT </td><td> 5微升5微升</td><td> 250 250 </td><td> SB9.5 </td><td>黄色</td><td> + </td></tr><tr><td> BCIP / INT原液</td><td> 7.5微升</td><td> 250 250 </td><td> SB9.5 </td><td>黄色</td><td> + </td></tr><tr><td>耐晒蓝NAMP </td><td> 5微升5微升</td><td> 250 250 </td><td> SB8.2 </td><td>蓝</td><td> ++ </td></tr><tr><td>耐晒蓝NABP </td><td> 20微升10微升</td><td> 1000 500 </td><td> SB8.2 </td><td>紫色</td><td> ++ </td></tr><tr><td>耐晒蓝NAGP </td><td> 10微升10微升</td><td> 500 500 </td><td> TT8.2 </td><td>绿色</td><td> + </td></tr><tr><td>耐晒大红NAMP </td><td> 1片</td><td> 1000 400 </td><td> TT8.2 </td><td>红</td><td> ++ </td></tr></tbody></table> 表1. 基材组合缩写:+++，强。 ++，介质; +，弱NBT，4-硝基蓝四唑氯化物; BCIP，5-溴-4-氯-3-吲哚基 - 磷酸酯; INT，2-（4-碘苯基）-3-（4-硝基苯基）-5-苯基四唑鎓氯化物; MAG，品红-PHOS，5-溴-6-氯-3-吲哚基 - 磷酸盐，NAMP，萘酚AS-MX磷酸; NABP，萘酚AS-BI磷酸盐; NAGP，萘酚AS-GR-磷酸盐; SB9.5，在pH9.5染色缓冲液; SB8.2，在pH 8.2染色缓冲液; TT8.2，在pH 8.2的Tris-吐温缓冲。

Watch this Scientific Journal Video about Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos at JoVE.com

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

我们描述在完整果蝇胚胎允许同时和特异性检测的三种不同的mRNA分布模式通过对比色沉淀物多靶生色整个安装原位杂交（MC-WISH）过程。

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

multi-target-chromogenic-whole-mount-situ-hybridization-for-comparing

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

11.4K 次观看.  Stockholm University. 我们描述在完整果蝇胚胎允许同时和特异性检测的三种不同的mRNA分布模式通过对比色沉淀物多靶生色整个安装原位杂交（MC-WISH）过程。 

视频: Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

科研

遗传学

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

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 ...

生物学

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 ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

发育生物学

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 ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

定量比较顺调节元件（CRE）的活动，在转基因果蝇</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

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 ...

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ within a given tissue remain limited. The EASI-FISH protocol described here has been adapted to detect the expression of dozens of genes in the intact adult Drosophila central nervous system (CNS) using commercially available reagents. This protocol includes a new gel formulation that enhances gel robustness, enabling multiple rounds of hybridization and allowing the embedding of multiple brains per gel. This improvement increases throughput, facilitates optimal comparison of experimental conditions, and reduces reagent costs. Additionally, by employing the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in specific neuronal or glial cell types. Notably, the high resolution achieved through expansion microscopy, combined with the sensitivity of the method, allows for the detection of single RNA transcripts. This approach effectively integrates high image quality with high throughput, making it a powerful tool for studying gene expression throughout the intact fly brain.

Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a robust technique that combines expansion microscopy with fluorescence in situ hybridization (FISH) to detect the expression of multiple genes in thick tissue. This protocol outlines recent advancements in the method and its adaptation for labeling the intact Drosophila central nervous system..

Understanding gene expression is essential for deciphering cellular functions. However, methods for analyzing the expression of numerous genes in situ ...

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高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

双整装原位杂交技术在早期鸡胚

斑马鱼整装高分辨率双荧光原位杂交

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输

全山原位杂交E8.5，E11.5小鼠胚胎

定量比较顺调节元件（CRE）的活动，在转基因果蝇

染色质免疫沉淀（ChIP）使用果蝇组织

全山RNA荧光

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

多目标显色全安装

摘要

探索更多视频

此视频中的章节

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

双整装原位杂交技术在早期鸡胚

斑马鱼整装高分辨率双荧光原位杂交

可视化 果蝇 胚胎中含脂质细胞器的细胞骨架依赖性运输

全山原位杂交E8.5，E11.5小鼠胚胎

定量比较顺调节元件（CRE）的活动，在转基因果蝇

染色质免疫沉淀（ChIP）使用果蝇组织

全山RNA荧光

Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization

可视化果蝇胚胎中含脂质细胞器的细胞骨架依赖性运输