我们感谢 Margo Herre 和 Leslie Vosshall 实验室分享他们对 埃及伊蚊 嗅觉附属物的原位杂交方案。这项工作得到了美国国立卫生研究院（National Institutes of Health）对C.J.P.（NIAID R01Al137078）的资助，对J.I.R.的HHMI Hanna Gray奖学金，对J.I.R.的约翰霍普金斯大学博士后加速器奖，以及约翰霍普金斯疟疾病研究所对J.I.R.的博士后奖学金。我们感谢约翰霍普金斯疟疾研究所和彭博慈善基金会的支持。

1. 材料的考虑和准备
<ol><li>确定组织全贴片或冷冻切片是否合适。该协议针对按蚊天线和上颌触诊中RNA的全位原位成像进行了优化，无需冷冻切片。如果样品厚度大于 5 mm，建议进行冷冻切片以使探针穿透。</li>
	<li>鉴定感兴趣的基因，并从合适的数据库中复制序列，包括内含子和外显子。将基因序列转录成RNA进行合成。</li>
	<li>确定是否从商业供应商处购买探针，或者是否在实验室中合成探针。 注:在这项研究中， 原位 探针是从商业供应商处购买的（见 材料表）。或者，它可以像先前报道的10一样合成。表 1描述了研究中使用的试剂。制备试剂所需的材料列在 材料表中。</li>
</ol>2.组织预固定
<ol><li>麻醉 10-15 只成年雌性或雄性 Coluzzii 按蚊 （菌株 N'Gousso），在出现后 5-10 天，用口吸器将它们收集到纸杯或塑料管中，并将它们放入装有冰块的桶中。当蚊子不动时，可以确认成功的冷诱导麻醉。</li>
	<li>用一把镊子从颈部区域取下头部，将它们斩首。用非惯用手抓住胸部，用惯用手握住的镊子将颈部与身体分开。将头部放入含有 500 μL 几丁质酶-胰凝蛋白酶二甲基亚砜缓冲液（CCD 缓冲液）的 1.5 mL 管中。 注意:冷冻动物会使触角变形;建议在冰上快速击倒。测试期间蚊子的年龄可能因项目而异。确认和协调它们的生理状态很重要，例如它们是以血为食、饥饿还是交配。在这项研究中，嗅觉附属物是从经过血液喂养和交配的蚊子身上取样的。</li>
	<li>在37°C的CCD缓冲液中将蚊子头在加热块上预热5分钟，然后将含有蚊子头的管转移到杂交炉中并在37°C下旋转。对于雌性 按蚊 触角，使用20分钟，雄性触角需要15分钟，而上颌触须需要更长的孵育时间（1小时）。</li>
	<li>将试管的全部内容物转移到解剖手表玻璃中。轻轻地将样品倒入手表（解剖）玻璃的凹陷处。如果蚊子头卡在试管内，请使用移液管将CCD缓冲液添加到试管中并冲洗掉蚊头。</li>
	<li>使用镊子小心地转移在孵育过程中分离的头部或任何触角/触须，并固定在 1 mL 的预固定剂中。 注意:剩余的CCD缓冲液可以在-20°C下保存，缓冲液可以重复使用3倍。如果预固定剂由于组织携带的 CCD 缓冲液而略呈褐色，请用另外 1 mL 固定剂代替。</li>
	<li>使用螺母器在4°C下将头旋转24小时。 注意:本研究中使用的悸动器的摇摆速度不可调;使用制造商设置的默认速度 （12 rpm）。</li>
</ol>3.组织解剖
<ol><li>用 1 mL 0.1% PBS-吐温在冰上冲洗头 4 次（每次洗涤 5 分钟）。为避免样品损失，请使用连接到凝胶上样吸头的移液管除去液体。 注意:两次快速洗涤，然后进行 10 分钟的长时间洗涤，最后可以执行快速洗涤，而不是步骤 3.1。</li>
	<li>将管中的头部转移到解剖手表玻璃中。轻轻将样品倒入手表玻璃的凹陷处。用 0.1% PBS-Tween 冲洗卡在管内的蚊子头。</li>
	<li>在解剖显微镜下，用锋利的镊子从头部取出感兴趣的组织（触角/触诊）。用镊子握住头部的后部，然后用另一根镊子从底部抓住一根天线。用不含RNAse的溶剂润湿的纸清洁镊子。使用相同的过程取下触诊。</li>
	<li>用镊子将触角和触诊转移到放置在冰上的无DNA/RNase空管中。将不同的组织部分分成预先标记的不同试管。</li>
	<li>在室温下，在含有甲醇（MeOH 80%）和二甲基亚砜（DMSO 20%）混合物的400μL溶剂中脱水组织1小时。 注意: 无需将纸巾放在 nutator 上;让它在室温下放在实验室工作台上的试管架上。建议使用新鲜的溶剂混合物。对于 500 μL 储备溶液，将 400 μL MeOH 与 100 μL DMSO 混合。</li>
	<li>用400μL无水（100%）甲醇代替脱水试剂，并在-20°C下使组织脱水过夜。让组织在重力作用下沉降，然后用移液管除去液体。 注意:样品可长时间脱水，最多 4 晚已测试过，没有信号丢失。</li>
</ol>4. 组织固定后
<ol><li>在冰上用分级的400μL MeOH / PBS-Tween的四步系列组织再水化10分钟。从 75% MeOH/25% PBS-吐温开始，然后是 50% MeOH/50% PBS-吐温，然后是 25% MeOH/75% PBS-吐温，最后是 100% PBS-吐温。 注意:可以使用带有微小开口的凝胶加载尖端从试管中取出缓冲液，以避免丢失组织。缓冲液去除可以在解剖显微镜下完成。</li>
	<li>在室温下用含有 0.1% 吐温-20 （PBS-T） 的 400 μL 磷酸盐缓冲盐水洗涤 10 分钟。将样品放在导向器上清洗。</li>
	<li>制备 20 μg/mL 蛋白酶-K 溶液，并在室温下在 400 μL 蛋白酶-K 溶液中孵育 30 分钟。 注:稀释 20 mg/mL 蛋白酶-K 储备液（1000x 储备溶液），即 2 μL 在 2 mL 0.1% PBS-吐温中。剩下的可以储存在-20°C冰箱中。</li>
	<li>用 400 μL 0.1% PBS-吐温洗涤 2 次 10 分钟，停止组织的酶消化。</li>
	<li>加入 400 μL 后固定剂，并在室温下孵育 20 分钟。用 400 μL 0.1% PBS-吐温洗涤 3 次，每次洗涤 15 分钟。</li>
</ol>5. 探针杂交
<ol><li>将组织在 400 μL 探针杂交缓冲液中孵育 5 分钟。通过轻轻移液确保组织完全浸没在缓冲液中。</li>
	<li>通过将探针杂交缓冲液的等分试样加热至37°C30分钟来准备下一步。</li>
	<li>除去缓冲液，用400μL预热的探针杂交缓冲液在37°C下预杂交30分钟。</li>
	<li>通过添加 8 pM 探针，为目标化学感觉受体（IR8a、IR76b、IR25a、IR41t.1、IR75d、IR7t、IR64a 或 Orco）制备探针溶液。将 8 μL 1 μM 探针原液加入 500 μL 预热探针杂交缓冲液中。</li>
	<li>取出预热的探针杂交缓冲液，并用 400 μL 加热的探针溶液代替。</li>
	<li>将组织在37°C下孵育两晚，放置在培养箱内并盖在盒子下的导向器上。</li>
</ol>6. 探针放大
<ol><li>将探针洗涤缓冲液加热至37°C，通过冲洗组织5次，每次洗涤10分钟，在37°C下用400μL探针洗涤缓冲液，在培养箱中交动，除去多余的探针溶液。 注意:步骤 6.4 的准备工作可能会开始。请参阅步骤 6.4 中的注释。</li>
	<li>在室温下用含有 10% 吐温-20 （SSCT） 的 400 μL 5x 盐水柠檬酸钠洗涤样品 2 次，每次洗涤 5 分钟。在工作台上将扩增缓冲液解冻至室温。</li>
	<li>通过在室温下与 400 μL 扩增缓冲液孵育 10 分钟来制备扩增组织。由于扩增缓冲液的粘度，组织可能不会完全浸没。通过轻轻移液将扩增缓冲液移液到组织下方并将缓冲液排出组织顶部直至浸没，从而混合。</li>
	<li>通过在95°C下加热6μL3μM原液90秒，并在黑暗的抽屉中冷却至室温30分钟，分别制备18pM发夹h1和18pM发夹h2。确保PCR管盖紧，以防止在热循环仪中加热时发夹蒸发。 注意:为了节省时间，可以在步骤6.4的第4个洗涤 步骤中启动步骤6.1。发夹应单独加热，防止交叉反应。在此步骤中，发夹不应用任何缓冲液稀释。发夹可以与探头一起从探头制造商处购买。</li>
	<li>将步骤6.3中添加的扩增缓冲液替换为含有加热的发夹（来自步骤6.4）的混合物以及100μL扩增缓冲液。典型反应将包含 6 μL h1、6 μL h2 和 100 μL 扩增缓冲液。 注意:发夹 h1 和 h2 可以在冷却至室温后合并，然后加入 100 μL 扩增缓冲液中。为了避免将组织样品从一个试管转移到另一个试管，可以去除步骤6.3中的扩增缓冲液，并用在扩增缓冲液中稀释的发夹混合物代替。</li>
	<li>孵育组织过夜，并在室温下使用章动器的默认速度在黑暗中章动。</li>
</ol>7. 安装组织样品
<ol><li>用 300 μL 5x SSCT（在吐温-20 中稀释的氯化钠-柠檬酸钠）稀释孵育组织中的扩增缓冲液。 注意:稀释有助于降低粘度，并更容易从组织中去除扩增缓冲液。我们使用 Triton-X 100 进行前固定，将 Tween-20 用于后固定步骤，因为它们作为透化细胞膜的试剂的作用存在差异。</li>
	<li>在室温下用 400 μL 5x SSCT 洗涤组织 5 次。 注意:如果需要，将组织暂时储存在4°C，直到准备好安装。我们在成像前不超过 2 天。</li>
	<li>在载玻片上制作 5 滴安装溶液。切割 200 μL 移液器吸头的吸头，使其变宽，然后将组织转移到新的载玻片上。</li>
	<li>用镊子抓住组织样本的基部，轻轻浸入并冲洗在一系列凝固溶液液滴中。注意不要在此步骤中折断组织。</li>
	<li>用安装液安装，放置盖玻片，并用指甲油密封。使用共聚焦显微镜对 原位 组织样品进行成像。</li>
</ol>

蚊子嗅觉附属物中的化学感应基因表达作图

<ol>
	<li>Konopka, J. K., 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=Olfaction+in+Anopheles+mosquitoes.">Olfaction in Anopheles mosquitoes.</a> Chem Senses. 46, (2021).</li><li>Raji, J. I., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+ionotropic+receptors+in+human+host-seeking+mosquitoes.">Chemosensory ionotropic receptors in human host-seeking mosquitoes.</a> Curr Opin Insect Sci. 54, 100967 (2022).</li><li>Young, A. P., Jackson, D. J., Wyeth, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technical+review+and+guide+to+RNA+fluorescence+in+situ+hybridization.">A technical review and guide to RNA fluorescence in situ hybridization.</a> PeerJ. 8, e8806 (2020).</li><li>Herre, M., 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=Non-canonical+odor+coding+in+the+mosquito.">Non-canonical odor coding in the mosquito.</a> Cell. 185 (17), 3104-3123.e28 (2022).</li><li>Raji, J. I., Konopka, J. K., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spatial+map+of+antennal-expressed+ionotropic+receptors+in+the+malaria+mosquito.">A spatial map of antennal-expressed ionotropic receptors in the malaria mosquito.</a> Cell Rep. 42 (2), 112101 (2023).</li><li>Choi, H. M. T., 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=Programmable+in+situ+amplification+for+multiplexed+imaging+of+mRNA+expression.">Programmable in situ amplification for multiplexed imaging of mRNA expression.</a> Nat Biotechnol. 28 (11), 1208-1212 (2010).</li><li>Choi, H. M. T., Beck, V. A., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+in+situ+hybridization+chain+reaction:+higher+gain,+lower+cost,+greater+durability.">Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability.</a> ACS Nano. 8 (5), 4284-4294 (2014).</li><li>Choi, H. M. T., 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=Third-generation+in+situ+hybridization+chain+reaction:+multiplexed,+quantitative,+sensitive,+versatile,+robust.">Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</a> Development. 145 (12), dev165753 (2018).</li><li>Schwarzkopf, M., 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=Hybridization+chain+reaction+enables+a+unified+approach+to+multiplexed,+quantitative,+high-resolution+immunohistochemistry+and+in+situ+hybridization.">Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization.</a> Development. 148 (22), dev199847 (2021).</li><li>Choi, H. M. T., 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=Mapping+a+multiplexed+zoo+of+mRNA+expression.">Mapping a multiplexed zoo of mRNA expression.</a> Development. 143 (19), 3632-3637 (2016).</li><li>Pitts, R. J., Derryberry, S. L., Zhang, Z., Zwiebel, L. J. <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+in+the+malaria+vector+mosquito+Anopheles+gambiae+tuned+to+amines+and+carboxylic+acids.">Variant ionotropic receptors in the malaria vector mosquito Anopheles gambiae tuned to amines and carboxylic acids.</a> Sci Rep. 7, 40297 (2017).</li><li>Rinker, D. C., Zhou, X., Pitts, R. J., Rokas, A., Zwiebel, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+transcriptome+profiles+of+anopheline+mosquitoes+reveal+human+host+olfactory+specialization+in+Anopheles+gambiae.">Antennal transcriptome profiles of anopheline mosquitoes reveal human host olfactory specialization in Anopheles gambiae.</a> BMC Genomics. 14, 749 (2013).</li><li>Maguire, S. E., Afify, A., Goff, L. A., Potter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-receptor-mediated+regulation+of+chemosensory+gene+expression+in+the+malaria+mosquito+Anopheles+gambiae.">Odorant-receptor-mediated regulation of chemosensory gene expression in the malaria mosquito Anopheles gambiae.</a> Cell Rep. 38 (10), 110494 (2022).</li><li>Athrey, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+gene+expression+in+olfactory+organs+of+the+anthropophilic+Anopheles+coluzzii+and+zoophilic+Anopheles+quadriannulatus.">Chemosensory gene expression in olfactory organs of the anthropophilic Anopheles coluzzii and zoophilic Anopheles quadriannulatus.</a> BMC Genomics. 18 (1), 751 (2017).</li><li>Task, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoreceptor+co-expression+in+Drosophila+melanogaster+olfactory+neurons.">Chemoreceptor co-expression in Drosophila melanogaster olfactory neurons.</a> eLife. 11, e72599 (2022).</li><li>Shah, S., 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=Single-molecule+RNA+detection+at+depth+by+hybridization+chain+reaction+and+tissue+hydrogel+embedding+and+clearing.">Single-molecule RNA detection at depth by hybridization chain reaction and tissue hydrogel embedding and clearing.</a> Development. 143 (15), 2862-2867 (2016).</li><li>Trivedi, V., Choi, H. M. T., Fraser, S. E., Pierce, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+quantitative+analysis+of+mRNA+expression+within+intact+vertebrate+embryos.">Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.</a> Development. 145 (1), dev156869 (2018).</li><li>Herre, M., Greppi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+in+situ+hybridization+and+immunohistochemistry+to+visualize+gene+expression+in+peripheral+chemosensory+tissues+of+mosquitoes.">RNA in situ hybridization and immunohistochemistry to visualize gene expression in peripheral chemosensory tissues of mosquitoes.</a> Cold Spring Harb Protoc. 2023 (1), 48-54 (2023).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+of+the+Year:+spatially+resolved+transcriptomics.">Method of the Year: spatially resolved transcriptomics.</a> Nat Methods. 18 (1), 9-14 (2021).</li></ol>

作者没有什么可透露的。

第三代杂交链式反应 （HCR） 因其灵敏度和稳健性而著称，可以可视化多个 RNA 靶标8。HCR WM-FISH已成功用于果蝇、鸡、小鼠和斑马鱼的胚胎以及线虫和斑马鱼的幼虫10,16,17。蚊子触角和上颌触诊通常容易出现高自发荧光和弱探针穿透，这在进行传统的全位安装方法时尤其具有挑战性。HCR方案中集成...

冈比亚按蚊等蚊媒依靠在其外周嗅觉附属物中表达的丰富的化学感觉基因库，在复杂的化学世界中茁壮成长，并识别来自人类宿主的行为相关气味，检测花蜜来源，并定位产卵部位1。蚊子触角和上颌触诊富含化学感觉基因，这些基因驱动这些嗅觉附属物的气味检测。三大类配体门控离子通道驱动蚊子嗅觉附属物的气味检测:气味受体 （OR），它与专性气味受体共受体 （Orco） 一起发挥作用;离子型受体 （IR），与一个或多个 IR 辅助受体（IR8a、IR25a 和 IR76b）相互作用;化学感觉味觉受体 （GR），它作为三种蛋白质的复合物来检测二氧化碳 （CO2）1,2。
RNA荧光原位杂交是检测内源性mRNA3表达的有力工具。通常，该方法使用荧光团标记的单链核酸探针，其序列与靶 mRNA 互补。荧光RNA探针与靶RNA的结合可以鉴定表达目标转录本的细胞。最近的进展现在能够检测整个蚊子组织中的转录本 4,5。第一代杂交链式反应（HCR）技术使用基于RNA的HCR扩增器;这在第二代方法中得到了改进，该方法将工程DNA用于HCR扩增6,7。这一升级使信号增加了 10 倍，生产成本大幅降低，并显著提高了试剂的耐久性 6,7。
在方案中，我们描述了第三代HCR全座RNA荧光原位杂交（HCR RNA WM-FISH）方法的利用，该方法旨在检测任何基因的空间定位和表达8,9。这种两步法首先利用对目标 mRNA 具有特异性的核酸探针，但其中也包含启动子识别序列;第二步利用荧光团标记的发夹，该发夹与引发剂序列结合以放大荧光信号（图1）。该方法还允许两个或多个 RNA 探针的多重检测和扩增探针信号，以促进 RNA 检测和定量8。可视化嗅觉附属物中表达的化学感觉基因的转录本丰度和 RNA 定位模式，为了解化学感觉基因功能和气味编码提供了第一线。

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hybridization chain reaction rna whole-mount fluorescence in situ hybridization of chemosensory genes in mosquito olfactory appendages

蚊子是致命疾病的有效载体，可以使用在其嗅觉附属物中表达的化学感觉受体在其化学环境中导航。了解化学感觉受体在外周嗅觉附属物中的空间组织方式，可以深入了解气味如何在蚊子嗅觉系统中编码，并为对抗蚊媒疾病的传播提供新方法。第三代杂交链式反应RNA全座荧光 原位 杂交（HCR RNA WM-FISH）的出现允许多个化学感觉基因的空间定位和同时表达谱。在这里，我们描述了在 按蚊 天线和上颌触诊上执行 HCR RNA WM-FISH 的逐步方法。我们通过检查离子型嗅觉受体的表达谱来研究该技术的敏感性。我们询问所描述的HCR WM-FISH技术是否适用于通过将RNA探针拴在三个光谱不同的荧光团上的多重研究。结果表明，HCR RNA WM-FISH对同时检测触角和上颌触诊嗅觉附肢中的多个化学感觉基因具有很强的敏感性。进一步的研究证明了HCR WM-FISH适用于双RNA和三RNA靶标的共表达谱。当应用修改时，这种技术可以适应于定位其他昆虫物种的嗅觉组织或其他附属物中感兴趣的基因。

按蚊天线中化学感觉基因的可靠检测 我们研究了HCR FISH方法（图1）检测蚊子嗅觉组织中化学感觉受体表达的灵敏度。在之前在雌性按蚊天线上报道的RNA转录数据的指导下，我们生成了靶向各种IR的探针。来自四项独立触角转录组研究的平均转录值显示，与共受体 Ir25a （197 RPKM） 和 Ir76b （193 RPKM） 相比，Ir41t.1 （11 RPKM）、Ir75d （...

Watch this Scientific Journal Video about Visualizing Olfactory Receptor Expression in Mosquitoes at JoVE.com

作者聚焦:可视化蚊子的嗅觉受体表达 

本文介绍了进行杂交链式反应 RNA 全座荧光 原位 杂交 （HCR RNA WM-FISH） 所需的方法和试剂，以揭示对蚊子触角和上颌触诊中化学感觉受体基因的空间和细胞分辨率的见解。

杂交链式反应 RNA全山荧光 蚊子嗅附物化学感觉基因 的原位 杂交

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory ...

我们小组对蚊子的化学感觉系统感兴趣。我们希望通过瞄准蚊子的味觉和嗅觉，我们可以阻止它找到我们并叮咬我们。杂交链式反应荧光原位杂交是最近的发展。该技术具有高度灵敏度、稳健性，并且能够对多个基因靶标进行多路复用，因此我们可以同时可视化这些基因在其天然环境中的表达和定位。特殊转录组学是空间生物学领域使用的前沿方法。该技术能够对数千个基因进行多路复用，您可以同时可视化这些基因的表达。我们的协议允许我们直接可视化天线中的哪些神经元表达 50 种左右可能的离子型受体中的任何一种。这为我们提供了一个基本的路线图，以指导我们理解这种经典因子受体如何介导行为。昆虫嗅觉器官（如触角）内的嗅觉神经元通常被埋在保护性几丁质壳内。这使得可视化这些神经元中的嗅觉受体表达非常具有挑战性。我们的方案结合使用几丁质酶处理和最先进的荧光和透视方案来克服这些挑战。

hybridization-chain-reaction-rna-whole-mount-fluorescence-situ

Research

JoVE Journal

Neuroscience

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages

1.1K 次观看.  Johns Hopkins University School of Medicine. 我们小组对蚊子的化学感觉系统感兴趣。我们希望通过瞄准蚊子的味觉和嗅觉，我们可以阻止它找到我们并叮咬我们。杂交链式反应荧光原位杂交是最近的发展。该技术具有高度灵敏度、稳健性，并且能够对多个基因靶标进行多路复用，因此我们可以同时可视化这些基因在其天然环境中的表达和定位。特殊转录组学是空间生物学领域使用的前沿方法。该技术能够对数千?...

视频: 作者聚焦:可视化蚊子的嗅觉受体表达 

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 ...

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

纤毛对小鼠的主要嗅觉系统整装标签

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

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 ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

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 ...

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

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

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

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

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 ...

杂交链式反应 RNA全山荧光 蚊子嗅附物化学感觉基因 的原位 杂交

杂交链式反应 RNA全山荧光蚊子嗅附物化学感觉基因的原位杂交