Name: localization of odorant receptor genes in locust antennae by rna in situ hybridization
Uploaded: 2017-07-13T13:00:00.000+00:00
Duration: 9 min 30 s
Description: Watch this Scientific Journal Video about Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization at JoVE.com

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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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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+odor+coding+in+the+Drosophila+antenna.">The molecular basis of odor coding in the Drosophila antenna.</a> Cell. 117 (7), 965-979 (2004).</li><li>Jones, W. D., Nguyen, T. A., Kloss, B., Lee, K. J., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+conservation+of+an+insect+odorant+receptor+gene+across+250+million+years+of+evolution.">Functional conservation of an insect odorant receptor gene across 250 million years of evolution.</a> Curr Biol. 15 (4), R119-R121 (2005).</li></ol>

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>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2&amp;times;TSINGKE&lt;sup&gt;TM&lt;/sup&gt; Master Mix</td><td>TSINGKE, China</td><td>TSE004</td><td></td></tr><tr><td>RNase-free H&lt;sub&gt;2&lt;/sub&gt;O</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>&lt;em&gt;Escherichia coli&lt;/em&gt; DH5&amp;alpha;</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 &amp;beta;-D-1-thiogalactopyranoside</td><td>Inalco, USA</td><td>1758-1400</td><td></td></tr><tr><td>5-bromo-4-chloro-3-indolyl-&amp;beta;-D-galactopyranoside</td><td>SBS Genetech, China</td><td>GX1-500</td><td></td></tr><tr><td>&lt;em&gt;Nco&lt;/em&gt; I</td><td>BioLabs, New England</td><td>R0193S</td><td></td></tr><tr><td>&lt;em&gt;Spe&lt;/em&gt; 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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1&amp;times;Tris-acetate-EDTA</td><td>Sigma, USA</td><td>V900483-1KG</td><td>0.04 mol/L Tris-Base</td></tr><tr><td>1&amp;times;Tris-acetate-EDTA</td><td>BEIJING CHEMICAL REGENTS COMPANY, China</td><td>10000292</td><td>0.12% acetic acid</td></tr><tr><td>1&amp;times;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>10 g/L NaCl</td></tr><tr><td>Luria-Bertani (LB) liquid medium</td><td>MERCK, Germany</td><td>VM335231</td><td>10 g/L Peptone from casein (Tryptone)</td></tr><tr><td>Luria-Bertani (LB) liquid medium</td><td>MERCK, Germany</td><td>VM361526</td><td>5 g/L Yeast extract</td></tr><tr><td>LB solid substrate plate</td><td>Sinopharm, China</td><td>10019392</td><td>10 g/L NaCl</td></tr><tr><td>LB solid substrate plate</td><td>MERCK, Germany</td><td>VM335231</td><td>10 g/L Peptone from casein (Tryptone)</td></tr><tr><td>LB solid substrate plate</td><td>MERCK, Germany</td><td>VM361526</td><td>5 g/L Yeast extract</td></tr><tr><td>LB solid substrate plate</td><td>WISENT ING, Canada</td><td>800-010-CG</td><td>15 g/L Agar Bacteriological Grade</td></tr><tr><td>10&amp;times;Phosphate-Buffered Saline (PBS, pH 7.1)</td><td>Sinopharm, China</td><td>10019392</td><td>8.5% NaCl</td></tr><tr><td>10&amp;times;Phosphate-Buffered Saline (PBS, pH 7.1)</td><td>Sigma, USA</td><td>V900041-500G</td><td>14 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>10&amp;times;Phosphate-Buffered Saline (PBS, pH 7.1)</td><td>Sigma, USA</td><td>V900268-500G</td><td>80 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>10&amp;times;Tris-Buffered Saline (TBS, pH 7.5)</td><td>Sigma, USA</td><td>V900483-1KG</td><td>1 M Tris-Base</td></tr><tr><td>10&amp;times;Tris-Buffered Saline (TBS, pH 7.5)</td><td>Sinopharm, China</td><td>10019392</td><td>1.5 M NaCl</td></tr><tr><td>Detection Buffer (DAP) &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; chromogenic detection pH 9.5&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TSA detection pH 8.0</td><td>Sigma, USA</td><td>V900483-1KG</td><td>100 mM Tris-Base</td></tr><tr><td>Detection Buffer (DAP) &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; chromogenic detection pH 9.5&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TSA detection pH 8.0</td><td>Sinopharm, China</td><td>10019392</td><td>100 mM NaCl</td></tr><tr><td>Detection Buffer (DAP) &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; chromogenic detection pH 9.5&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; TSA detection pH 8.0</td><td>Sigma, USA</td><td>V900020-500G</td><td>50 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>20&amp;times;saline-sodium citrate (pH 7.0)</td><td>Sinopharm, China</td><td>10019392</td><td>3 M NaCl</td></tr><tr><td>20&amp;times;saline-sodium citrate (pH 7.0)</td><td>Sigma, USA</td><td>V900095-500G</td><td>0.3 M Na-Citrate</td></tr><tr><td>4% paraformaldehyde solution (PFA, pH 9.5)</td><td>Sigma, USA</td><td>V900894-100G</td><td>4% paraformaldehyde</td></tr><tr><td>Sodium Carbonate Buffer</td><td>Sigma, USA</td><td>V900182-500G</td><td>0.1 M NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium Carbonate Buffer (pH 10.2)</td><td>Sigma, USA</td><td>V900182-500G</td><td>80 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium Carbonate Buffer (pH 10.2)</td><td>Sigma, USA</td><td>S7795-500G</td><td>120 mM Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Formamide Solution (pH 10.2)</td><td>MPBIO, USA</td><td>FORMD002</td><td>50% Deionized Formamide</td></tr><tr><td>Formamide Solution (pH 10.2)</td><td></td><td></td><td>5&amp;times;saline-sodium citrate</td></tr><tr><td>Blocking Buffer in TBS</td><td>Roche, Switzerland</td><td>11175041910</td><td>1% Blot</td></tr><tr><td>Blocking Buffer in TBS</td><td>AMRESCO, USA</td><td>0694-500ML</td><td>0.03% Triton X-100</td></tr><tr><td>Blocking Buffer in TBS</td><td></td><td></td><td>1&amp;times;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 TBS</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 TBS</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&amp;times;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 &amp;micro;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&#039;-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&#039;-phenylanilide phosphate (10 mg/mL)</td></tr><tr><td>2-hydroxy-3-naphtoic acid-2&#039;-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 (25 mg/mL)</td></tr><tr><td>2-hydroxy-3-naphtoic acid-2&#039;-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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instrument&lt;/strong&gt;</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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed in sensory hairs called sensilla, which cover the surface of olfactory organs. The surface of each sensillum is covered with tiny pores, through which odorants pass and dissolve in a fluid called sensillum lymph, which bathes the sensory dendrites of the OSNs housed in a given sensillum. The OSN dendrites express odorant receptor (OR) proteins, which in insects function as odor-gated ion channels4, 5. The interaction of odorants with ORs either increases or decreases the basal firing rate of the OSN. This neuronal activity in the form of action potentials embodies the first representation of the quality, intensity, and temporal characteristics of the odorant6, 7.
Given the easy access to these sensory hairs, it is possible to perform extracellular recordings from single OSNs by introducing a recording electrode into the sensillum lymph, while the reference electrode is placed in the lymph of the eye or body of the insect. In Drosophila, sensilla house between one and four OSNs, but each OSN typically displays a characteristic spike amplitude. Spike sorting techniques make it possible to assign spiking responses to individual OSNs. This single sensillum recording (SSR) technique monitors the difference in potential between the sensillum lymph and the reference electrode as electrical spikes that are generated by the receptor activity on OSNs1, 2, 8. Changes in the number of spikes in response to the odorant represent the cellular basis of odor coding in insects. Here, we describe the preparation method currently used in our lab to perform SSR on Drosophila melanogaster and Anopheles gambiae, and show representative traces induced by the odorants in a sensillum-specific manner.

Single Sensillum Recordings in the Insects Drosophila melanogaster and Anopheles gambiae

Electrophysiological responses of olfactory sensory neurons to odorants can be measured in insects using single sensillum recordings. In this video article we will demonstrate how to perform single sensillum recordings in the antennae of the vinegar fly (Drosophila melanogaster) and the maxillary palps of the malaria mosquito (Anopheles gambiae).

在昆虫单感器记录果蝇按蚊</em

The sense of smell is essential for insects to find foods, mates, predators, and oviposition sites3. Insect olfactory sensory neurons (OSNs) are enclosed ...

科研

神经科学

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 ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

果蝇分子和免疫组化分析制备周围嗅组织的研制

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

发育生物学

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

在自主机器人以昆虫触角电位传感器的嗅觉搜索

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Electroantennography-based Bio-hybrid Odor-detecting Drone using Silkmoth Antennae for Odor Source Localization

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their applicability in environmental and security monitoring and search-and-rescue operations. Small drones with commercial metal-oxide-semiconductor (MOX) gas sensors have been developed for odor source localization; however, their real-time-odor-detection performance has proven inadequate. However, biosensing technologies based on insect olfactory systems exhibit relatively high sensitivity, selectivity, and real-time response with respect to odorant molecules compared to commercial MOX gas sensors. In such devices, excised insect antennae function as portable odorant biosensor elements and have been found to deliver excellent sensing performance. This study presents experimental protocols for odorant-molecule detection in the air using a small autonomous bio-hybrid drone based on a mountable electroantennography (EAG) device incorporating silkmoth antennae.
We developed a mountable EAG device including sensing/processing parts with a Wi-Fi module. The device was equipped with a simple sensor enclosure to enhance the sensor directivity. Thus, odor source localization was conducted using the spiral-surge algorithm, which does not assume an upwind direction. The experimental bio-hybrid odor-detecting drone identified real-time odorant-concentration differences in a pseudo-open environment (outside a wind tunnel) and localized the source. The developed drone and associated system can serve as an efficient odorant molecule-detection tool and a suitable flight platform for developing odor source localization algorithms owing to its high programmability.

This study introduces experimental protocols for a bio-hybrid odor-detecting drone based on silkmoth antennae. The operation of an experimental electroantennogram device with silkmoth antennae is presented, in addition to the structure of a bio-hybrid drone designed for odor source localization using the spiral-surge algorithm.

基于电子电图的生物混合气味检测无人机使用丝质天线进行气味源本地化

Small drones with chemical or biosensor devices that can detect airborne odorant molecules have attracted considerable attention owing to their ...

工程学

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 ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

使用单一感器录音来检测臭虫的嗅觉神经元的反应，以信息化学物质

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

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. ...

Single Sensillum Recordings for Locust Palp Sensilla Basiconica

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially for the detection of non-volatile chemical cues through sensilla chaetica (previously named terminal sensilla or crested sensilla). There is now increasing evidence that these palps also have an olfactory function. An odorant receptor (LmigOR2) and an odorant-binding protein (LmigOBP1) have been localized in the neurons and accessory cells, respectively, in the sensilla basiconica of the palps. Single sensillum recording (SSR) is used for recording the responses of odorant receptor neurons, which is an effective method for screening active ligands on specific odorant receptors. SSR is used in functional studies of odorant receptors in palp sensilla. The structure of the sensilla basiconica located on the dome of the palps differs somewhat from the structure of those on the antennae. Therefore, when performing an SSR elicited by odorants, some specific advice may be helpful for obtaining optimum results. In this paper, a detailed and highly effective protocol for an SSR from insect palp sensilla basiconica is introduced.

This paper describes a detailed and highly effective protocol for single sensillum recordings from the sensilla basiconica on the palps of insect mouthparts.

蝗虫下颚触角 Basiconica 单触角记录

The palps of locust mouthparts are considered to be conventional gustatory organs that play an important role in a locust&#39;s food selection, especially ...

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

通过RNA定位蝗虫天线中的气味受体基因

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果蝇分子和免疫组化分析制备周围嗅组织的研制

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基于电子电图的生物混合气味检测无人机使用丝质天线进行气味源本地化

在脑切片检测轴突本地化mRNAs的高空间分辨率

使用单一感器录音来检测臭虫的嗅觉神经元的反应，以信息化学物质

实时 cAMP 检测气味受体激活的活细胞测量

蝗虫下颚触角 Basiconica 单触角记录

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization