这项工作得到国家自然科学基金资助项目（No.1472037）的支持。本文中提及的商品名称或商业产品仅用于提供具体信息，并不意味着推荐。

注意:为了限制RNA降解，请使用湿式高压灭菌蒸馏水（121℃，60分钟）和湿式高压釜材料制备溶液。 RNA ISH反义和检测探针的制备<ol><li> 靶基因扩增和纯化 <ol><li>首先，从含有LmigOR1全长cDNA的质粒与Taq DNA聚合酶一起产生A. migratoria OR1 （ LmigOR1 ，GenBank:JQ766965）的387bp双链片段，该Taq DNA聚合酶在片段的两端添加腺嘌呤。 <ol><li>使用100μL最终反应体积，含有50μL的2x反应混合物，4μL有义和反义引物（ 表1 ），1μL质粒模板和41μL无RNase的H 2 O.使用以下PCR方案:94℃5分钟，然后是94℃30秒，55℃30秒和72℃1分钟的30个循环，72℃10分钟。 </li><li>重复这些步骤以产生LmigOR2的1,303bp双链片段（GenBank:JQ766966）和Lmigorco （GenBank:JN989549）的1,251bp双链片段。这些实验中使用的引物列于表1 。 </li><li>在1％Tris-acetate-EDTA缓冲液中的1.2％琼脂糖凝胶上运行PCR产物，并使用溴化乙锭染色观察。 </li></ol></li><li>使用凝胶提取试剂盒提取这些PCR产物。 <ol><li>电泳后，从凝胶中切下DNA条带，将凝胶切片置于1.5 mL管中。然后，每0.1克凝胶切片加入100μL结合缓冲液（缓冲液PN）。涡旋并在50℃下孵育直到凝胶切片完全溶解。 </li><li>将CA2吸附柱插入收集管，加入500μL平衡缓冲液（Buffer BL）。这提高了二氧化硅膜的吸收能力和稳定性。 CENTRI以12,400 x g的速度。for 1分钟。 </li><li>将溶解的凝胶混合物转移到吸附柱组件，并在室温下孵育2分钟。然后，以12,400 xg离心管1分钟。放弃流通并将吸附柱重新插入收集管。 </li><li>加入600μL洗液（乙醇加缓冲液PW）两次。以12,400 xg离心1分钟。放弃流通并将吸附柱重新插入收集管。 </li><li>将收集管清空并以12,400×g离心柱组件2分钟以允许蒸发残留的乙醇。 </li><li>小心地将吸附塔转移到干净的1.5 mL离心管中。将沉淀物干燥5-10分钟，并将DNA重新溶解在合适体积（ 例如 30μL）无核酸酶的水中。 </li><li>在室温下孵育2分钟。以12,400 xg离心2分钟。丢弃吸附柱，并将DNA储存在4°C或-20°C。 </li></ol></li></ol></li><li> 构建重组质粒 <ol><li>单独结扎LmigOR1，LmigOR2的1303 bp片段和LmigORco的1251 bp的片段插入到包含用于T7（上游）和邻近于使用T4DNA连接酶的插入DNA SP6（下游）的RNA聚合酶启动子一T载体的387 bp片段。准备以下10μL反应:5μL2x连接缓冲液，1μLT载体，3μL（〜100 ng）插入的基因DNA和1μLT4 DNA连接酶，并在4°C孵育O / N。 </li><li>添加含有LmigOR1的387 bp片段，LmigOR2的1303 bp片段和LmigORco的1251 bp的片段分别导入在无菌的1.5mL管主管大肠杆菌 DH5α细胞的50微升的各重组质粒的5μL。 <ol><li>轻轻混合并将管置入冰中30分钟，然后在42℃孵育90秒C。将它们转移回冰中3分钟。 </li><li>在每个管中加入450μL无氨苄青霉素的Luria-Bertani（LB）液体培养基，并在振荡器中以37℃将其孵育1小时，以恢复大肠杆菌 DH5α。 </li><li>取每个转化体的100μL等分试样，并使用它们接种含有50μg/ mL氨苄青霉素，24μg/ mL异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）和40μg/ mL 5-溴-4 - 氯-3-吲哚基-β-D-吡喃半乳糖苷（X-gal）。 </li><li>在37℃恒温培养箱中孵育这些板18小时。使用蓝白色筛选方法筛选靶标重组质粒。使用Sanger测序序列目标重组质粒，并鉴定三个OR基因的插入方向。 </li></ol></li></ol></li><li> 选择限制性内切核酸酶线性化重组质粒 <ol><li>使用限制性内切核酸酶帽子不仅在载体的多克隆位点消化，而且不切割插入DNA。在该实验中，将所有3个基因以相对于载体的方向插入到T载体中。 </li><li>使用的Nco I限制性核酸内切酶进行线性化含有LmigOR1的387 bp片段，LmigOR2的1303 bp片段和LmigORco的1251 bp片段，以产生反义探针的重组质粒。使用Sp e I限制性内切核酸酶产生感应探针。 注意:如果插入方向与载体方向相符，则选择T7末端的唯一限制性位点，使重组质粒线性化，并使用SP6 RNA聚合酶制备反义探针。选择SP6末端的独特的限制性位点使重组质粒线性化，并使用T7 RNA聚合酶制备感官探针。否则，如果插入方向不对应于t选择载体方向，然后从SP6末端选择一个独特的限制性位点，使重组质粒线性化，并使用T7 RNA聚合酶制备反义探针。选择T7末端的唯一限制性位点使重组质粒线性化，并使用SP6 RNA聚合酶制备有义探针。 </li></ol></li><li> 纯化线性重组质粒 <ol><li>消化20微克每片含LmigOR1的387 bp片段，LmigOR2的1303 bp的片段，并使用的Nco I限制性核酸内切酶在包含10x缓冲液10微升100微升反应LmigORco的1251 bp的片段，40μL三个重组质粒的0.5μg/μL质粒， 3μLNco I和47μL无RNA酶的H 2 O，然后在37℃下孵育3小时。 </li><li>类似地，消化20μg这三种重组质粒中的每一种唱Spe I限制性内切核酸酶。 </li><li>使用如1.1.2所述的凝胶提取试剂盒纯化这些消化的质粒。通过分光光度法测定这些提取的线性化重组质粒的浓度，调整为0.5μg/μL。 </li></ol></li><li> 准备反义和有义探针 <ol><li>使用T7 / SP6 RNA转录系统产生反义和感官探针。使用SP6 RNA聚合酶转录用于DIG-和生物素标记的反义探针的双链RNA用于这三种OR基因，使用线性化重组质粒作为体外模板。 T7 RNA聚合酶用于产生有义探针。在20μL终体积中进行反应，含有2μL10x NTP混合物，2μL10X转录缓冲液，1μLRNA酶抑制剂，2μLT7或SP6 RNA聚合酶，4μL0.5μg/μL线性化质粒和9μL无RNase的H 2 O，其次为在37℃孵育3小时。 </li><li>在37℃下，用1μLDNA酶将DIG-和生物素标记的反义和感应探针孵育30分钟。加入2.5μL5M LiCl和75μL无水乙醇，然后在-70℃下将反应物温育30分钟。 </li><li>在4℃下以15,000xg离心30分钟。仔细倾倒上清液。加入150μL70％乙醇洗涤沉淀剂。通过向无菌蒸馏水（263.2mL）中加入95％乙醇（736.8mL）制备70％乙醇（1L）。 </li><li>在4℃下以15,000xg离心30分钟并在室温下空气干燥沉淀5-10分钟。加入25μL无RNase的H 2 O溶解RNA反义和感应探针。 </li><li>如果插入的基因的长度长于1kb，随后通过在碳酸氢盐 - 碳酸盐缓冲溶液中温育，将片段RNA反义和有义探针平均长度达〜300bp。以50μL的最终体积进行反应，c的RNA探针的ontaining 25μL和碳酸氢盐碳酸盐缓冲溶液的25μL（80mM的碳酸氢钠 ，120毫的Na 2 CO 3，pH值10.2）在60℃下。所需的水解时间由先前公布的公式17给出 。 </li><li>加入5μL的10％乙酸停止反应。在本实验中，分别将LmigOR2和LmigORco反义和感应探针片段分离 24和23分钟。 t =（L o -L f ）/ kXL o XL f L o =以kb为单位的初始片段长度 L f =以kb为单位的最终片段长度 k =水解速率常数，约0.11 kb -1 min -1 t =水解时间（分钟） </li><li>最后加入250μL杂交缓冲液，储存于-80°C。 </li></ol></li></ol> 2.制备低温恒温器部分<ol><li>预冷自由子ng切片机至-24℃。 </li><li>选择新的蜕皮成虫蝗虫，并且具有完整的天线。使用无菌剃刀将天线切成2-3毫米。 </li><li>将OCT化合物置于冷冻切片机支架上，并将一个或两个样品水平放置在化合物上。然后，将支架转移到-24℃的冷冻切片机中以平衡直到化合物冻结。取出持有人，并用少许复合物覆盖样品。将支架置于-24°C的冷冻切片机中至少10分钟（ 图1 ）。 注意:避免在化合物中吸入气泡。 </li><li>将样品固定在冷冻切片机中，然后在-24℃下将冷冻样品切成12μm厚的切片。 </li><li>将切片在载玻片（25 x 75 mm;不含核酸酶）上逐个安装切片，风干10分钟。 </li></ol> 3.固定部分<ol><li>准备低温恒温器后切片，将载玻片置于塑料容器（约100 x 40 x 80 mm）中，并通过将载玻片在4％多聚甲醛溶液（PFA）中4℃孵育30分钟来固定组织。 </li><li>在1X磷酸盐缓冲盐水（PBS）中洗涤载玻片1分钟。 </li><li>为了消除碱性蛋白质，将载玻片转移到0.2M HCl 10分钟。 </li><li>为了消除核酸的表面蛋白，用1％Triton X-100将载玻片转移到1XPBS 2分钟。 </li><li>在1x PBS中洗涤载玻片两次30秒。 </li><li>最后，在4℃下将载玻片在甲酰胺溶液中冲洗10分钟。 </li></ol>杂交<ol><li> 准备反义和感应探针 <ol><li>使用杂交缓冲液稀释1.5毫升无核酸酶管中的RNA反义或感应探针。在本实验中，对于所有反义和有义探针（ LmigOR1，LmigOR2和LmigOrco ），加入1μL探针至99μL杂交缓冲液。通常使用1:100稀释的反义探针可以使用该方案产生显着的正信号和低背景。 </li><li>对于显色检测，分别稀释DIG标记的探针。 </li><li>对于TSA检测，稀释DIG标记LmigOR1与生物素标记的探针LmigOrco或DIG标记的LmigOR2与生物素标记探针LmigOR1一起在杂交缓冲液中。 </li><li>在65℃下将稀释液加热10分钟，并将其放在冰上至少5分钟。 </li></ol></li><li> 杂交 <ol><li>排出载玻片，并将100μL稀释的反义和感应探针（步骤4.1）加入到组织切片中。然后，将盖玻片（24毫米×50毫米，无核酸酶）放置在组织切片上。 </li><li>将覆盖的水平放置在潮湿的箱子中（〜300 x 180 x 50 mm 3 ; 图2 ），并孵化at 55°C 22 h。将甲酰胺溶液或1X PBS加入到盒子的底部，以保持环境潮湿，但不要将载玻片浸没在液体中。 </li></ol></li><li> 洗涤和阻塞。 <ol><li>杂交后，小心取出盖玻片。在0.1x盐水柠檬酸钠（SSC）中，60℃下将载玻片两次洗涤30分钟。 注意:洗涤意味着将滑块放入滑动架中，然后将其放入塑料容器中，并在摇臂上轻轻搅动（约50 rpm; 图2 ）。 </li><li>在1x Tris缓冲盐水（TBS）中冲洗载玻片30秒。 </li><li>在每个载玻片上补充有0.03％Triton X-100的TBS中加入1mL的1％封闭试剂，并孵育30分钟。然后，丢弃阻塞解决方案。 </li></ol></li><li> 免疫组化。 <ol><li>对于显色检测，使用TBS中的封闭试剂稀释750单位（U）/ mL抗地高辛AP-缀合的抗体dy至1.5 U / mL AP溶液。每个覆盖（24 mm x 50 mm）幻灯片添加100μLAP溶液。 </li><li>对于TSA检测，使用TBS中的封闭试剂稀释到AP / HRP溶液中750U / mL抗地高辛AP-缀合的抗体和抗生物素链霉亲和素HRP-缀合的抗体。每个覆盖（24 mm x 50 mm）幻灯片添加100μLAP / HRP溶液。 </li><li>在潮湿的箱子（ 图2 ）中孵育玻片在37℃下孵育60分钟。使用甲酰胺溶液或1X PBS保持箱子潮湿但不潮湿。 </li></ol></li></ol>染色<ol><li>仔细取出盖玻片。在补充有0.05％Tween-20的1×TBS中洗涤载玻片3次5分钟。在DAP缓冲液（显色检测:pH9.5; TSA检测:pH8.0）中冲洗载玻片5分钟。 </li><li> 染色（显色检测） <ol><li>加入100μLNBT（375μg/ mL）/ BCIP（188μg/ mL）底物溶液（稀释于DAP;pH 9.5）。小心地将玻片（24 x 50毫米）放在幻灯片上。 </li><li>在含有底物溶液的潮湿箱中，将载玻片在37℃下孵育10分钟--O / N。 注意:通过在显微镜下不时查看幻灯片来检查发展。 </li><li>当开发准备就绪时，通过将载玻片转移到水中来停止反应。 </li></ol></li><li> 染色（TSA检测） <ol><li>使用注射器从注射器过滤器（0.22μm; 图2 ）移动HNPP（100μg/ mL）/ Fast Red（250μg/ mL）底物。 </li><li>在每片玻片上加盖100μL的HNPP / Fast Red底物，盖玻片（24 x 50 mm）。在室温下用HNPP / Fast Red底物孵育载玻片30分钟。 </li><li>仔细取出盖玻片，并在补充有0.05％Tween-20的1X TBS中洗涤载玻片三次5分钟。 </li><li>使用100μLTSA底物/覆盖（24 x 50 mm 2 ）幻灯片。使用TSA底物孵育载玻片10分钟。 </li><li>仔细取出盖玻片，并在补充有0.05％Tween-20的1x TBS中洗涤载玻片三次，持续5分钟。 </li></ol></li><li>将载玻片嵌入PBS /甘油（1:3）中。 注意:完成这些程序后，应尽快观察幻灯片，因为荧光信号会快速熄灭。 </li></ol>观察<ol><li> 显色检测 <ol><li>使用光学显微镜观察组织切片。选择10X，20X和40X物镜观察显色检测结果。 </li><li>使用DP-BSW软件分析结果并捕获图像。 </li></ol></li><li> TSA检测 <ol><li>使用共焦显微镜观察组织切片。应在543nm光下观察DIG标记的基因，呈现红色，生物素标记的基因应为在488nm光下观察，呈现绿色。当它们出现时，它们呈现黄色。 </li><li>选择20X和40X物镜分别观察TSA检测结果。 </li><li>使用FV1000软件分析结果并捕获图像。 </li></ol></li></ol>

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S., Rzhetsky, A., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+map+of+olfactory+receptor+expression+in+the+Drosophila+antenna.">A spatial map of olfactory receptor expression in the Drosophila antenna.</a> Cell. 96 (5), 725-736 (1999).</li><li>Yang, Y., Krieger, J., Zhang, L., Breer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+olfactory+co-receptor+Orco+from+the+migratory+locust+(Locusta+migratoria)+and+the+desert+locust+(Schistocerca+gregaria):+identification+and+expression+pattern.">The olfactory co-receptor Orco from the migratory locust (Locusta migratoria) and the desert locust (Schistocerca gregaria): identification and expression pattern.</a> Int J Biol Sci. 8 (2), 159-170 (2012).</li><li>Schultze, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+co-expression+pattern+of+odorant+binding+proteins+and+olfactory+receptors+identify+distinct+trichoid+sensilla+on+the+antenna+of+the+malaria+mosquito+Anopheles+gambiae.">The co-expression pattern of odorant binding proteins and olfactory receptors identify distinct trichoid sensilla on the antenna of the malaria mosquito Anopheles gambiae.</a> PLoS One. 8 (7), e69412 (2013).</li><li>Xu, H., Guo, M., Yang, Y., You, Y., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+two+novel+odorant+receptors+in+the+locust+(Locusta+migratoria).">Differential expression of two novel odorant receptors in the locust (Locusta migratoria).</a> BMC Neurosci. 14, 50 (2013).</li><li>Saina, M., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+olfactory+receptor+expression+and+localization+in+Drosophila.">Visualizing olfactory receptor expression and localization in Drosophila.</a> Methods Mol Biol. 1003, 211-228 (2013).</li><li>Guo, M., Krieger, J., Gro&#223;e-Wilde, E., Mi&#223;bach, C., Zhang, L., Breer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+ionotropic+receptors+are+expressed+in+olfactory+sensory+neurons+of+coeloconic+sensilla+on+the+antenna+of+the+desert+locust+(Schistocerca+gregaria).">Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria).</a> Int J Biol Sci. 10 (1), 1-14 (2013).</li><li>You, Y., Smith, D. P., Lv, M., Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+broadly+tuned+odorant+receptor+in+neurons+of+trichoid+sensilla+in+locust,+Locusta+migratoria.">A broadly tuned odorant receptor in neurons of trichoid sensilla in locust, Locusta migratoria.</a> Insect Biochem Mol Biol. 79, 66-72 (2016).</li><li>Jiang, X., Pregitzer, P., Grosse-Wilde, E., Breer, H., Krieger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+two+"Sensory+Neuron+Membrane+Proteins"+(SNMPs)+of+the+desert+locust,+Schistocerca+gregaria+(Orthoptera:+Acrididae).">Identification and characterization of two "Sensory Neuron Membrane Proteins" (SNMPs) of the desert locust, Schistocerca gregaria (Orthoptera: Acrididae).</a> J Insect Sci. 16 (1), 33 (2016).</li><li>Angerer, L. M., Angerer, R. C., Wilkinson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ.+hybridization+to+cellular+RNA+with+radiolabelled+RNA+probes.">In situ. hybridization to cellular RNA with radiolabelled RNA probes.</a> In situ hybridization. , 2 (1992).</li><li>Komminoth, P., Merk, F. B., Leav, I., Wolfe, H. J., Roth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+35S-+and+digoxigenin-labeled+RNA+and+oligonucleotide+probes+for+in+situ+hybridization.+Expression+of+mRNA+of+the+seminal+vesicle+secretion+protein+II+and+androgen+receptor+genes+in+the+rat+prostate.">Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.</a> Histochemistry. 98 (4), 217-228 (1992).</li><li>Leary, J. J., Brigati, D. J., Ward, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+colorimetric+method+for+visualizing+biotin-labeled+DNA+probes+hybridized+to+DNA+or+RNA+immobilized+on+nitrocellulose:+Bio-blots.">Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.</a> Proc Natl Acad Sci U S A. 80 (13), 4045-4049 (1983).</li><li>Hallem, E. A., Ho, M. G., Carlson, J. 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作者没有披露或任何其他利益冲突。

这是很难执行RNA ISH本地化或昆虫触角基因，因为的表达水平或基因除了ORCO，是非常低的和保存的组织学切片的昆虫触角是非常困难的。另外TSA检测也很棘手。为解决这些问题，应采取以下措施。天线选自具有薄且软的触角表皮的新蜕皮成虫蝗虫，其保持其在幻灯片上的形态。将冷冻样品切成12μm厚的切片。使用高灵敏度和特异性的生物素和DIG标记的探针。洗涤剂如Tween-20和Triton X-100用于在许多步骤中降低背景。在TSA检测中，相对于另一个低表达基因的高表达基因应用生物素-16-UTP进行标记。应尽快观察幻灯片，因为荧光信号会快速熄灭。 DIG- a第二生物素标记的探针具有较长的货架寿命的优点，更高的信噪比和更好的蜂窝分辨率高于放射性探针18,19。本文提出的方案具有优于整体原位杂交的优点。该方案很容易识别基因在细胞水平上的定位，但是不能容易地用于研究基因分布模式，其更容易在全原位杂交中进行。 为了鉴定OR基因，采用了两种方法。一个是连续切片中的显色检测，另一个是在一个部分中的荧光检测。 ORCO是共表达与特定的或作为ORN标记10，20，21。均位于表达LmigOR1或LmigOR2的细胞到LmigORco表达细胞的簇， 将其明确地验证为OR（ 图4和图6 ）。使用相同的方法，我们发现LmigOR1和LmigOR2在一个感官中不共表达。这两种方法的结果是肯定的。 该协议还成功地用于定位LmigORco，SgreORco，SgreIR8a和SgreIR25a在刺槐天线10，14。最近LmigOR3使用相同的方案15定位于L. migratoria中的毛状感觉神经元。该方案用于定位两种感觉神经膜蛋白 SgreSNMP1和SgreSNMP2在S. gregaria 16的天线中。因此，该方案可靠地定位蝗虫天线中的化学感应相关基因，不仅验证了这些基因候选基因，但也将这些基因定位在不同类型感觉器中的特定细胞。 总之，RNA ISH的这种高效的方案被具体描述为在昆虫天线中定位OR基因以及低水平表达的其他基因。

作为最重要的化学感觉器官的昆虫天线被许多由嗅觉受体神经元（ORN）支配的头发状结构（称为感觉）覆盖。昆虫ORNs的膜，气味受体（ORS），一种类型的包含七个跨膜结构域的蛋白质，与共同受体（ORCO）以形成杂聚体表示其功能如同一个加臭剂门控离子通道1，2，3。不同的OR响应化学化合物4，5，6的不同组合。 蝗虫（ Locusta migratoria ）主要依靠嗅觉信号触发重要行为7 。蝗虫OR是理解分子嗅觉机制的关键因素。将特定的蝗虫OR基因定位到a的神经元通过RNA的形态特异性感觉类型原位杂交（RNA ISH）是探索ORs功能的第一步。 RNA ISH使用标记的互补RNA探针来测量和定位组织，细胞或整个原位部分的特异性RNA序列，从而提供对生理过程和疾病发病机理的见解。地高辛标记（DIG标记）和生物素标记的RNA探针已被广泛用于RNA杂交。用地高辛-11-UTP或生物素-16-UTP进行RNA标记可以通过SP6和T7 RNA聚合酶的体外转录来制备。 DIG-和生物素标记的RNA探针具有以下优点:非放射性;安全;稳定;高度敏感;高度特异性并且使用PCR和体外转录容易产生。 DIG-和生物素标记的RNA探针可以发色和荧光检测。 DIG标记的RNA探针可以用抗地高辛碱碱检测使用光学显微镜或2位可以用高度敏感的化学发光底物硝基蓝四唑氯化物/ 5-溴-4-氯-3-吲哚基 - 磷酸酯甲苯胺盐（NBT / BCIP）可视化的ne磷酸酶（AP）共轭抗体使用共聚焦显微镜与4-氯-2-甲基苯并噻唑半氯化锌盐（Fast Red）偶合的3-羟基-3-萘甲酸-2&#39;-苯基苯胺磷酸盐（HNPP）。可以用抗生物素链霉亲和素马萝卜过氧化物酶（HRP） - 共轭抗体检测生物素标记的RNA探针，该抗体可以使用共聚焦显微镜用荧光素 - 酪酰胺显色。因此，可以进行双色荧光原位杂交以使用DIG-和生物素标记的RNA探针在一个切片中检测两个靶基因。 具有DIG和/或生物素标记的探针的RNA ISH已被成功地用于定位嗅相关基因，如OR ，离子型受体，加味剂结合蛋白和感觉神经元的膜蛋白，在昆虫天线，但不限于， 黑腹果蝇 ， 冈比亚按蚊 ，L.飞蝗和沙漠蝗虫Schistocera蝗 8，9，10，11，12，13，14，15，16。然而，为昆虫ORs进行RNA ISH时有两个实质性的挑战:（1）OR基因（ ORco除外）以低水平表达，仅在少数细胞中表达，使信号检测非常困难，（2）保存昆虫组织组织学，使得形态被保留并且背景噪声低，可能是具有挑战性的。在本文中，详细和有效的方案描述了用于在昆虫中定位OR基因的RNA ISH提出了天线，包括显色和Tyramide信号放大（TSA）检测。

<table>
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2&#215;TSINGKETM Master Mix</td>
			<td>TSINGKE, China</td>
			<td>TSE004</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free H2O</td>
			<td>TIANGEN, China</td>
			<td>RT121-02</td>
			<td></td>
		</tr>
		<tr>
			<td>REGULAR AGAROSE G-10</td>
			<td>BIOWEST, SPAIN</td>
			<td>91622</td>
			<td></td>
		</tr>
		<tr>
			<td>Binding buffer</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance buffer</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash solution</td>
			<td>TIANgel Midi Purification Kit, TIANGEN, China</td>
			<td>DP209-02</td>
			<td></td>
		</tr>
		<tr>
			<td>T Vector</td>
			<td>Promega, USA</td>
			<td>A362A</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Promega, USA</td>
			<td>M180A</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945;</td>
			<td>TIANGEN, China</td>
			<td>CB101</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma, USA</td>
			<td>A-6140</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside</td>
			<td>Inalco, USA</td>
			<td>1758-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside</td>
			<td>SBS Genetech, China</td>
			<td>GX1-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nco I</td>
			<td>BioLabs, New England</td>
			<td>R0193S</td>
			<td></td>
		</tr>
		<tr>
			<td>Spe I</td>
			<td>BioLabs, New England</td>
			<td>R0133M</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG RNA Labeling Kit</td>
			<td>Roche, Switzerland</td>
			<td>11175025910</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin RNA Labeling Kit</td>
			<td>Roche, Switzerland</td>
			<td>11685597910</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>DIG RNA Labeling Kit, Roche, Switzerland</td>
			<td>11175025910</td>
			<td></td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Sinopharm, China</td>
			<td>10012718</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sinopharm, China</td>
			<td>10009257</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>BEIJING CHEMICAL REGENTS COMPANY, China</td>
			<td>10000292</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura Finetek Europe, Zoeterwoude, Netherlands</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides</td>
			<td>TINA JIN HAO YANG BIOLOGCAL MANUFACTURE CO., LTE, China</td>
			<td>FISH0010</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sinopharm, China</td>
			<td>80070591</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex</td>
			<td>Millipore, USA</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>AMRESCO, USA</td>
			<td>0777-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitroblue tetrazolium chloride / 5-bromo-4-chloro-3-indolyl-phosphate toluidine salt</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm, China</td>
			<td>10010618</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>0.04mol/L Tris-Base</td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>BEIJING CHEMICAL REGENTS COMPANY, China</td>
			<td>10000292</td>
			<td>0.12%acetic acid</td>
		</tr>
		<tr>
			<td>1&#215;Tris-acetate-EDTA</td>
			<td>Sigma, USA</td>
			<td>03677</td>
			<td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>10g/L NaCl</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>MERCK, Germany</td>
			<td>VM335231</td>
			<td>10g/L Peptone from casein (Tryptone)</td>
		</tr>
		<tr>
			<td>Luria-Bertani (LB) liquid medium</td>
			<td>MERCK, Germany</td>
			<td>VM361526</td>
			<td>5g/L Yeast extract</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>10g/L NaCl</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>MERCK, Germany</td>
			<td>VM335231</td>
			<td>10g/L Peptone from casein (Tryptone)</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>MERCK, Germany</td>
			<td>VM361526</td>
			<td>5g/L Yeast extract</td>
		</tr>
		<tr>
			<td>LB solid substrate plate</td>
			<td>WISENT ING, Canada</td>
			<td>800-010-CG</td>
			<td>15g/L Agar Bacteriological Grade</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>8.5%NaCl</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sigma, USA</td>
			<td>V900041-500G</td>
			<td>14mM KH2PO4</td>
		</tr>
		<tr>
			<td>10&#215;phosphate buffer saline (pH7.1)</td>
			<td>Sigma, USA</td>
			<td>V900268-500G</td>
			<td>80mM Na2HPO4</td>
		</tr>
		<tr>
			<td>10&#215;Tris buffered saline (pH7.5)</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>1M Tris-Base</td>
		</tr>
		<tr>
			<td>10&#215;Tris buffered saline (pH7.5)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>1.5M NaCl</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sigma, USA</td>
			<td>V900483-1KG</td>
			<td>100mM Tris-Base</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>100mM NaCl</td>
		</tr>
		<tr>
			<td>Detection Buffer (DAP) &#160;&#160;&#160;&#160;&#160; chromogenic detection pH9.5&#160;&#160;&#160;&#160;&#160;&#160; TSA detection pH8.0</td>
			<td>Sigma, USA</td>
			<td>V900020-500G</td>
			<td>50mM MgCl2&#183;6H2O</td>
		</tr>
		<tr>
			<td>20&#215;saline-sodium citrate (pH7.0)</td>
			<td>Sinopharm, China</td>
			<td>10019392</td>
			<td>3M NaCl</td>
		</tr>
		<tr>
			<td>20&#215;saline-sodium citrate (pH7.0)</td>
			<td>Sigma, USA</td>
			<td>V900095-500G</td>
			<td>0.3M Na-Citrate</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution (pH9.5)</td>
			<td>Sigma, USA</td>
			<td>V900894-100G</td>
			<td>4% paraformaldehyde</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde solution (pH9.5)</td>
			<td>Sigma, USA</td>
			<td>V900182-500G</td>
			<td>0.1M NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Buffer (pH10.2)</td>
			<td>Sigma, USA</td>
			<td>V900182-500G</td>
			<td>80mM NaHCO3</td>
		</tr>
		<tr>
			<td>Sodium Carbonate Buffer (pH10.2)</td>
			<td>Sigma, USA</td>
			<td>S7795-500G</td>
			<td>120mM Na2CO3</td>
		</tr>
		<tr>
			<td>Formamide Solution (pH10.2)</td>
			<td>MPBIO, USA</td>
			<td>FORMD002</td>
			<td>50% Deionized Formamide</td>
		</tr>
		<tr>
			<td>Formamide Solution (pH10.2)</td>
			<td></td>
			<td></td>
			<td>5&#215;saline-sodium citrate</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1% Blot</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td>AMRESCO, USA</td>
			<td>0694-500ML</td>
			<td>0.03% Triton X-100</td>
		</tr>
		<tr>
			<td>Blocking Buffer in Tris buffered saline</td>
			<td></td>
			<td></td>
			<td>1&#215;Tris buffered saline</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase solution</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase solution</td>
			<td></td>
			<td></td>
			<td>Blocking Buffer in Tris buffered saline</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td>Roche, Switzerland</td>
			<td>11175041910</td>
			<td>1.5 U/ml anti-digoxigenin alkaline phosphatase conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>1% anti-biotin streptavidin horse radish peroxidase- conjugated antibody</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase/ horse radish peroxidase solution</td>
			<td></td>
			<td></td>
			<td>Blocking Buffer in Tris buffered saline</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>MPBIO, USA</td>
			<td>FORMD002</td>
			<td>50% Deionized Formamide</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td></td>
			<td></td>
			<td>2&#215;saline-sodium citrate</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>Sigma, USA</td>
			<td>D8906-50G</td>
			<td>10% dextran sulphate</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>invitrogen, USA</td>
			<td>AM7119</td>
			<td>20 &#181;g/ml yeast t-RNA</td>
		</tr>
		<tr>
			<td>Hybridization Buffer</td>
			<td>Sigma, USA</td>
			<td>D3159-10G</td>
			<td>0.2 mg/ml herring sperm DNA</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td>Roche, Switzerland</td>
			<td>11758888001</td>
			<td>1% 2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate (10mg/ml)</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td>Roche, Switzerland</td>
			<td>11758888001</td>
			<td>1% 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt (25mg/ml)</td>
		</tr>
		<tr>
			<td>2-hydroxy-3-naphtoic acid-2&#39;-phenylanilide phosphate/ 4-chloro-2-methylbenzenediazonium hemi-zinc chloride salt substrate</td>
			<td></td>
			<td></td>
			<td>Detection Buffer</td>
		</tr>
		<tr>
			<td>Tyramide signal amplification substrate</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>2% fluorescein-tyramides</td>
		</tr>
		<tr>
			<td>Tyramide signal amplification substrate</td>
			<td>TSA kit, Perkin Elmer, USA</td>
			<td>NEL701A001KT</td>
			<td>Amplification Diluent</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Instrument</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Leica, Nussloch, Germany</td>
			<td>Jung CM300 cryostat</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo SCIENTIFIC, USA</td>
			<td>NANODROP 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>Olympus IX71microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus, Tokyo, Japan</td>
			<td>Olympus BX45 confocal microscope</td>
			<td></td>
		</tr>
	</tbody>
</table>

localization of odorant receptor genes in locust antennae by rna in situ hybridization

昆虫已经发展了复杂的嗅觉接收系统来感测外源化学信号。这些化学信号由嗅觉受体神经元（ORNs）转导，其位于天线的头发状结构（称为化学感光体）中。在ORN的膜上，认为气味受体（ORs）参与气味编码。因此，能够识别定位于ORN的基因是识别OR基因所必需的，为进一步的功能性原位研究提供了基础。昆虫天线中特异性OR的RNA表达水平非常低，用于组织学的保存昆虫组织具有挑战性。因此，使用RNA 原位杂交难以将OR定位到特定类型的感觉器。在本文中，介绍了详细和高效的RNA 原位杂交方案，特别是对昆虫的低表达OR基因。另外还有一个具体的OR 基因哇通过使用共表达受体基因Orco进行双色荧光原位杂交实验鉴定 。

通过显色检测，每个成年天线部分的小部分触角细胞由DIG标记的LmigOR1和LmigOR2反义探针表示（ 图3 ）。连续切片上的RNA ISH定位LmigOR1和LmigOR2显示表达两个基因的触角细胞位于表达LmigORco的 ORN簇中，表明推定的LmigOR1和LmigOR2实际上在ORN中表达（ 图4a -4d ）。有时，标记的树状样结构可视化（ 图4e -4f ）。 LmigOR1和LmigOR2均位于基底细胞感觉器中的神经元（ 图5a-5b ），但它们在单个感觉体中不共表达，表明它们存在于不同的碱性<html>敏感敏感亚型（ 图5c- 5d ）。 在TSA检测中，双色荧光原位杂交显示LmigOR1表达细胞（红色）位于表达LmigORco （绿色）的ORN簇，表明推测的LmigOR1在ORN中表达（ 图6a ）。 图6a中的盒装区域的近视图 如图6b所示。 LmigOR1表达神经元（绿色， 图7a ）和LmigOR2表达神经元（红色， 图7b ）位于不同的基底感觉神经元亚型（ 图7c ）。 <html> 4 / 55924fig1.jpg"/&gt; 图1:RNA 原位杂交的昆虫天线切片的制备。 （ a ）冷冻切片机; （ bc ）将样品嵌入冷冻的OCT化合物中。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55924/55924fig2.jpg" /> 图2:RNA 原位杂交的 实验项目 。 （ a ）滑动架和塑料容器。 （ b ）湿箱。 （ c ）摇杆。 （ d ）膜过滤器。 （ e ）共焦显微镜。<html> .jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。 <img alt="图3" class="xfigimg" src="/files/ftp_upload/55924/55924fig3.jpg" /> 图3:嗅觉器官中LmigOR1和LmigOR2的细胞定位。 （ a ）蝗虫触角段中LmigOR1表达细胞的概述。 （ b ）蝗虫触角段中LmigOR2表达细胞的概述。箭头表示表达LmigOR1 （ a ）和LmigOR2 （ b ）的细胞。刻度棒=100μm（a和b）。数据来自Xu 等人 12 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <html> "&gt; <img alt="图4" class="xfigimg" src="/files/ftp_upload/55924/55924fig4.jpg" /> 图4:通过显色检测表达LmigOR的天体细胞的神经元识别。 （ ab ）蝗虫天线连续部分的LmigOR1 （ a ）和LmigORco （ b ）反义探针的标记模式。 （ cd ）蝗虫天线连续部分的Lmigorco （ c ）和LmigOR2 （ d ）反义探针的标记模式。 （ ef ）偶尔标记的树枝状结构（由红色箭头表示）的图示。刻度棒=50μm（ad）; 20μm（e和f）。数据来自Xu 等人 12 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> nt"fo:keep-together.within-page ="1"&gt; <img alt="图5" class="xfigimg" src="/files/ftp_upload/55924/55924fig5.jpg" /> 图5: LmigOR1和LmigOR2在位于基底神经敏感的ORN中表达。 （ ab ）Basiconic Sensillum容纳表达LmigOR1 （ a ）和LmigOR2 （ b ）的ORN 。 （ cd ）在连续切片（cd）上验证了触角ORN的不同子集中LmigOR1和LmigOR2的表达。箭头表示表达LmigOR1 （a，c）和LmigOR2 （b，d）的触角细胞。 Ba:basiconic sensillum。刻度棒=20μm（a和b）; 50μm（c和d）。数据来自Xu 等人 12 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图6:TSA检测表达LmigOR1的天门细胞的神经元识别。 （ a ）在纵向天线部分进行双色原位杂交，以说明LmigOR1 （Red）和Lmigorco （Green）的表达。表达Lmigorco的细胞簇中LmigOR1表达细胞的定位证实了其神经认知 。 （ b ）关闭一个盒装区域的视图。用箭头表示偶尔标记的树状结构。圆形区域表示ORNs簇表达Lmigorco并且共享相同的感官 。刻度棒=50μm（a）; 20μm（b）。数据来自Xu 等人 12 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig6large.jpg" target="_blank">请坐</a>舔这里查看这个数字的较大版本。 <img alt="图7" class="xfigimg" src="/files/ftp_upload/55924/55924fig7.jpg" /> 图7: LmigOR1和LmigOR2位于不同的Basiconic Sensilla ORNs通过TSA检测。使用检测系统显现荧光信号，通过绿色荧光（ a ）表明LmigOR1标记的神经元和红色荧光（ b ）的LmigOR2阳性细胞在不同的基底感觉亚型（ c ）中表达。箭头表示表达LmigOR1和LmigOR2的天线细胞。刻度棒=20μm。数据来自Xu 等人 12 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55924/55924fig7large.jpg" target="_blank">请点击这里查看</a>这个数字的较大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 名称 </td><td> 序列 </td></tr><tr><td> Lmig OR1探针</td><td> 5&#39;-AAGGGGTGGGAGACGGCCTG-3&#39; </td></tr><tr><td> Lmig OR1-probe-as </td><td> 5&#39;-CAGCTCCTCCCCAACGACAGC-3&#39; </td></tr><tr><td> Lmig OR2探针</td><td> 5&#39;-ATGGGTGAGCGTGGAGAGGC-3&#39; </td></tr><tr><td> Lmig OR2-probe-as </td><td> 5&#39;-GGTCATCGCTGTGGACGTGG-3&#39; </td></tr><tr><td> Lmig Orco探测器</td><td> 5&#39;-CTCGTCTGACAGCGTAACTCAC-3&#39; </td></tr><tr><td> Lmig Orco-probe-as </td><td> 5&#39;-AAGACGCAGAAGAGGAAGACCT-3&#39; </td></tr></tbody></table> 表1:PCR引物的序列。

Watch this Scientific Journal Video about Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization at JoVE.com

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

本文描述了一种详细和高效的RNA 原位杂交方案，特别是对于低水平表达的气味受体（OR）基因以及其他基因，在使用地高辛（DIG）标记或生物素标记的探针的昆虫天线中。

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

localization-odorant-receptor-genes-locust-antennae-rna-situ

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

8.0K 次观看.  China Agricultural University. 本文描述了一种详细和高效的RNA 原位杂交方案，特别是对于低水平表达的气味受体（OR）基因以及其他基因，在使用地高辛（DIG）标记或生物素标记的探针的昆虫天线中。 

视频: Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.