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

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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方案中集成的信号放大步骤减少了这些缺点，该步骤提高了信噪比，并允许检测嗅觉化学感受器mRNA转录本（图2）。HCR 方案设计确保起始探针与扩增聚合物相连，从而允许以低信号对 RNA 靶标进行成像。在多路复用装置中，不同的正交HCR放大器被拴在光谱上独特的荧光团上。这种方法对于避免来自相邻成像通道的光谱渗漏至关重要。
为了优化用于蚊子附属物的HCR方法，我们进行了一些观察。HCR放大器是光敏的，应始终存放在-20°C冰箱的盒子中。根据我们的经验，组织在CCD缓冲液、蛋白酶K中广泛孵育或在多聚甲醛中短时间固定可能会导致在洗涤步骤中触角或触须断裂。触角和上颌触诊也应始终被基部抓住，以避免断裂。在适应该方案的早期阶段，我们在一系列缓冲液交换（步骤5和6）中始终丢失组织。为了尽量减少这种损失，在实验开始时将组织数量增加一倍，在显微镜下进行洗涤步骤和缓冲液交换，并使用宽度不超过0.5mm的凝胶上样尖端从组织中去除缓冲液。
HCR WM-FISH在冈比亚按蚊5,15和埃及伊蚊4,18蚊媒的外周嗅觉附属物中取得了成功，并且该方案可以进一步优化并适应其他昆虫组织部位或不同的动物。制造商设计的原始方案未将孵育纳入CCD缓冲液中。该步骤被整合到方案中，以消化和透化几丁质角质层。我们将探针组的孵育时间延长了两个晚上，以便有更多时间进行探针穿透，这是对制造商方案的修改，该方案建议孵育时间为 16 小时，适用于载玻片上的通用样品。针对其他昆虫组织部分优化该方案需要改变CCD缓冲液中的孵育时间;具有较厚角质层的外周组织部分可能需要延长孵育时间。蛋白酶K的浓度也必须通过实验调整，同时使该方案适应动物的不同组织或发育阶段。当处理厚度大于 5 mm 的组织时，探头穿透成为一项挑战。在这种情况下，需要额外的努力来进行组织冷冻切片或进一步改变本研究中描述的方案。
与空间转录组学相比，HCR WM-FISH仅限于一次只能同时可视化几个基因靶标，空间转录组学可能允许同时对数千个基因进行成像19。RNA探针的商业合成成本高昂;另一种方法是在实验室中生产探头和放大器，但这对于大多数团队来说可能具有挑战性，并且劳动密集型且耗时。该方案还受到RNA探针穿透整个厚样品时遇到的困难的限制，这可能需要改变方案（例如，步骤2组织制备）或对完整组织进行切片。如果此处描述的 HCR WM-FISH 方法未能检测到嗅觉附属物中的 RNA 转录本，则该基因可能仅在少数细胞中表达，并且需要对组织进行广泛的调查，可能未转录，或者转录的水平可能低于该方法的检测限。可以进行定量RT-PCR或RNA seq来验证这些发现。

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。

<table><tbody><tr><td>Amplification buffer</td><td>Molecular Instruments</td><td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td><td>50 mL</td></tr><tr><td>Calcium Chloride (CaCl2) 1M&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>21115-100ML</td><td></td></tr><tr><td>Chitinase</td><td>Sigma-Aldrich</td><td>C6137-50UN</td><td></td></tr><tr><td>Chymotrypsin</td><td>Sigma-Aldrich</td><td>CHY5S-10VL&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>472301</td><td></td></tr><tr><td>Eppendorf tube</td><td>VWR</td><td>20901-551</td><td>1.5 mL</td></tr><tr><td>Forceps</td><td>Dumont</td><td>11251</td><td>Number 5</td></tr><tr><td>Gel loading tip</td><td>Costar</td><td>4853</td><td>1-200 &amp;micro;L tip</td></tr><tr><td>Hairpins&amp;nbsp;</td><td>Molecular Instruments</td><td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td><td>h1 and h2 initiator splits</td></tr><tr><td>HEPES (1M)</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>IR25a probe</td><td>Molecular Instruments</td><td>Probe Set ID: PRK149&amp;nbsp;</td><td>AGAP010272</td></tr><tr><td>IR41t.1 probe</td><td>Molecular Instruments</td><td>&amp;nbsp;Probe Set ID: PRK978</td><td>AGAP004432</td></tr><tr><td>IR64a probe</td><td>Molecular Instruments</td><td>Probe Set ID: PRK700&amp;nbsp;</td><td>AGAP004923</td></tr><tr><td>IR75d probe</td><td>Molecular Instruments</td><td>Probe Set ID: PRK976</td><td>AGAP004969</td></tr><tr><td>IR76b probe</td><td>Molecular Instruments</td><td>Probe Set ID: PRI998</td><td>AGAP011968</td></tr><tr><td>IR7t probe</td><td>Molecular Instruments</td><td>Probe Set ID: PRL355</td><td>AGAP002763</td></tr><tr><td>IR8a probe</td><td>Molecular Instruments</td><td>Probe Set ID: PRK150</td><td>AGAP010411</td></tr><tr><td>LoBind Tubes</td><td>VWR</td><td>80077-236</td><td>0.5 mL DNA/RNA LoBind Tubes</td></tr><tr><td>Magnessium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;) 1M</td><td>Thermo Fisher</td><td>AM9530G</td><td></td></tr><tr><td>Methanol</td><td>Fisher&amp;nbsp;</td><td>A412-500</td><td></td></tr><tr><td>Nuclease-free water</td><td>Thermo Fisher</td><td>43-879-36</td><td></td></tr><tr><td>Nutator</td><td>Denville Scientific</td><td>Model 135</td><td>3-D Mini rocker</td></tr><tr><td>Orco probe</td><td>Molecular Instruments</td><td>Probe set ID PRD954</td><td>AGAP002560</td></tr><tr><td>Paraformaldehyde (20% )</td><td>Electron Microscopy Services&amp;nbsp;</td><td>15713-S</td><td></td></tr><tr><td>Phosphate Buffered Saline (10X PBS)</td><td>Thermo Fisher</td><td>AM9625</td><td></td></tr><tr><td>Probe hybridization buffer</td><td>Molecular Instruments</td><td>https://www.molecularinstruments.com/</td><td>50 mL</td></tr><tr><td>Probe wash buffer</td><td>Molecular Instruments</td><td>Molecular Instruments, Inc. | In Situ Hybridization + Immunofluorescence</td><td>100 mL</td></tr><tr><td>Proteinase-K</td><td>Thermo Fisher</td><td>AM2548</td><td></td></tr><tr><td>Saline-Sodium Citrate (SSC) 20x&amp;nbsp;</td><td>Thermo Fisher</td><td>15-557-044</td><td></td></tr><tr><td>SlowFade Diamond</td><td>Thermo Fisher&amp;nbsp;</td><td>S36972</td><td>mounting solution</td></tr><tr><td>Sodium Chloride (NaCl) 5M</td><td>Invitrogen</td><td>AM9760G</td><td></td></tr><tr><td>Triton X-100&amp;nbsp; (10%)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>93443</td><td></td></tr><tr><td>Tween-20 (10% )</td><td>Teknova</td><td>T0027</td><td></td></tr><tr><td>Watch glass</td><td>Carolina</td><td>742300</td><td>&amp;nbsp;1 5/8&quot; square; transparent</td></tr></tbody></table>

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 （12 RPKM） 和 Ir7t （13 RPKM） 在天线中的丰度较低5,11,12,13,14。我们生成了靶向 Ir41t.1、Ir75d 和 Ir7t 的探针。IR64a （31 RPKM） 也被靶向，因为它的转录水平比目标候选基因中的最低转录值丰富约 3 倍。这里必须注意的是，转录组学研究的RPKM值代表了整个天线转录本的大量测量。因此，可能是只有少数神经元高度表达 IR 基因转录本，也可能是 IR 基因在许多细胞中低表达。因此，RPKM值不一定对应于神经元内IR的丰度水平。此外，HCR方法提供的信号放大也可能使得使用该方法根据荧光信号准确测量神经元转录水平变得困难。我们最近的出版物更详细地讨论了这些概念5.数据表明，HCR WM-FISH对检测触角组织中的mRNA转录物高度敏感，如图2所示。
化学感觉附属物中不同RNA靶标的多重共标记 为了检查RNA靶标的共定位，我们生成了与不同荧光团偶联的探针。靶向气味受体共受体基因 （Orco） 和最广泛表达的离子型受体共受体基因 （Ir25a） 转录本的双重原位杂交揭示了这些不同的化学感觉受体家族在天线（图 3A）和上颌触诊（图 3B）的细胞群子集中的共定位。我们还研究了三个 IR 共受体基因（Ir8a、Ir25a 和 Ir76b）转录本的共定位。共定位模式表明 Ir76b 阳性细胞表达 Ir25a，而 Ir8a 阳性细胞部分与 Ir76b 和 Ir25a 共定位（图 4）。IR 共受体的共表达分析证明了使用 HCR WM-FISH 进行多重研究的稳健性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65933/65933fig01.jpg"> 图1： 原位 杂交链式反应示意图。 该方法分两步工作：检测和扩增。用于 原位 杂交链反应的探针组包括分裂引发剂和针对RNA靶标的核酸序列。可以设计多对探针组来杂交RNA靶标中的多个区域;这定义了检测步骤。扩增步骤需要荧光团标记的发夹（h1 和 h2）特异性结合到探针组偶联的引发剂。结合后，荧光团标记的RNA信号通过荧光团标记的发夹的重复结合而扩增。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65933/65933fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65933/65933fig02.jpg"> 图 2：IR 化学感觉基因的 RNA 定位。 雌性 按蚊 触角的 HCR WM-FISH 靶向 IR。RNA 探针包括 （A） IR41t.1、（B） IR75d、（C） IR7t 和 （D） IR64a。白色虚线框中的插图是来自组织的放大图像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65933/65933fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65933/65933fig03.jpg"> 图 3：触角和上颌触诊中化学感觉基因的双重原位共定位。 雌性按蚊的 （A） 触角和 （B） 上颌触诊中表达 Ir25a（绿色）和 Orco（洋红色）的细胞的共聚焦 Z 堆栈图像。白色虚线框中的插图是来自组织的放大图像。刻度为10μm。 图3B已从15修改而来。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65933/65933fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65933/65933fig04.jpg"> 图 4：绘制多个 RNA 靶标的空间共定位图。 原 位 图像显示了蚊子天线中三种 IR 共受体 IR25a（品红色）、IR8a（绿色）和 IR76b（蓝色）的共定位模式。白色箭头指向表达三种 IR 共受体（IR25a、IR8a 和 IR76b）的细胞。刻度为20μm。此数字已从5 修改而来。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65933/65933fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
表1： 原位 杂交链式反应试剂。 进行杂交链式反应所需的试剂，全座荧光 原位 杂交。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65933/Table%201_RE.xlsx" target="_blank">请按此下载此表格。</a>

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

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

1.2K 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 ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

免疫与感染

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

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 ...

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

生物学

荧光 原位 杂交研究蚊子精子发生

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

作者聚焦:全安装荧光原位杂交研究按蚊精子发生  

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

全安装荧光 原 位杂交研究 按 蚊精子发生

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

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

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双荧光原位杂交食脑切片

结合亲脂性染料，原位杂交，免疫组织化学和组织学

杂交

荧

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

嗅觉受体神经元在全山免疫标记

通过RNA定位蝗虫天线中的气味受体基因

全山RNA荧光

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生

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

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摘要

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双荧光原位杂交食脑切片

结合亲脂性染料，原位杂交，免疫组织化学和组织学

杂交

荧

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

嗅觉受体神经元在全山免疫标记

通过RNA定位蝗虫天线中的气味受体基因

全山RNA荧光

全安装荧光 原 位杂交研究 按 蚊精子发生

全安装荧光 原 位杂交研究 按 蚊精子发生

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

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

全安装荧光原位杂交研究按蚊精子发生

全安装荧光原位杂交研究按蚊精子发生