这项工作得到了科学和高等教育部对SARS-CoV-2研究的支持，并得到了国家科学中心的赠款（向K.P.和UMO-2018/30/E/NZ1/00874赠款给A.K.-P.）。

1. 缓冲区准备
<ol><li>对于 500 mL 的 2 倍 PHEM 缓冲器，将 18.14 克管道拉辛 -N，N+-二（2-乙烷硫酸）（PIPES），6.5 克 4-（2-羟基苯甲酸） 1-管道丙烷硫酸（HEPES）、3.8克乙二醇四乙酸（EGTA）和0.99克硫酸镁（MgSO4）。用蒸馏水（dH2O）使体积达到~400毫升，搅拌，然后使用10M氢氧化钾（KOH）或氢氧化钠（NAOH）将pH调整到7.0。使最终音量达到 500 mL，然后拆分为 50 mL 的别名。储存在-20°C，直到需要。 注:缓冲区在 pH 达到 7.0 之前不会清晰。</li>
	<li>准备 37% 的库存溶液，其中不含甲醛 （PFA）。对于 50 毫升，将 18.5 克 PFA 和 35 毫升 dH2O 混合在玻璃瓶中。将瓶子放在带加热的磁性搅拌器上。加入 900 μL 的 1 M KOH 或 NaOH，搅拌，直到解决方案变得清晰。允许冷却并转移到50mL圆锥离心机管:充值至50毫升与dH2O。解决方案可存储在 -20 °C，直到需要为止。 注意:甲醛应在戴防护手套和实验室外套时用烟熏罩处理。</li>
	<li>准备固定缓冲区（3.7%PFA缓冲与PHEM）。对于 50 mL，在 50 mL 锥形离心机管中组合 5 mL 的 37% PFA 解决方案、25 mL 的 2 倍 PHEM 缓冲器和 20 毫升 dH2O。储存在-20°C，直到需要。 注意:解冻后，缓冲区可存储在 4 °C 以至 3 个月之久。</li>
	<li>准备PBST（0.1%补间-20在1x磷酸盐缓冲盐水[PBS]）。对于 50 mL，添加 50 μL 的 100% 补间-20 至 50 mL 的 1x PBS，并很好地混合。 注:解决方案可在室温下存储 （RT）。</li>
	<li>通过将 MeOH/PBST 组合在 3:1、1:1 和 1:3 的比例（每份最终体积为 100 mL;参见 表 1）来准备补液缓冲器。 注意:MEOH是非常有毒和易燃的。因为它可以损害视神经，它应该在烟雾罩处理，避免任何明火。由于混合MeOH和PBST会产生外热反应，因此将冰上的溶液组合在一起。</li>
	<li>对于 1000 毫升的 20x SSC，将 175.3 克氯化钠 （NaCl） 和 88.2 克柠檬酸钠混合在烧瓶中，并加满 800 毫升的 dH2O. 搅拌至溶解，并使用 NaOH 将 pH 调整为 7.2。将 dH2O 添加到总体积为 1000 mL 中，然后通过 0.22 μm 滤镜将高压灭菌器或滤芯过滤到自封瓶中。</li>
	<li>对于 50 毫升的 5 倍 SSCT，将 20 倍 SSC 缓冲区的 12.5 mL 与 37.5 mL 的 dH2O 组合在一起，并添加 50 μL 的 100% Tween-20。混合好。</li>
	<li>对于 50 mL 的 50% 5x SSCT/50% PBST，将 25 mL 的 5 倍 SSCT 与 25 mL 的 PBST 组合在一起。</li>
	<li>对于 2x SSC 的 50 毫升，将 5 毫升的 10 倍 SSC 缓冲区与 45 毫升的 dH2O. 混合好。 注意:所有SSC缓冲区应存储在黑暗中的RT。</li>
</ol>2. 目标定义、探头和放大器
<ol><li>使用制造商网站上可用的工具来设计放大器和探头。确保探针与SARS-CoV-2 N基因的反向DNA链（补充图1）相辅相成。</li>
	<li>确定最佳探头集大小（一组 20 个探头足以可视化）。</li>
	<li>设置用于目标RNA检测的放大器。<ol>
<li>使用标有亚历克萨氟647的HCR放大器B1。 注:HCR 放大器包括可转移的 HCR 发夹 h1 和 h2。使用此协议可以设计多路复用实验。如果计划这样做，请选择不同的HCR放大器（B1，B2，...），以便在同一样本中对每个目标RNA进行成像（例如，目标1的放大器B1，目标2的放大器B2，...）。</li>
	</ol>
</li>
</ol>3. 细胞培养和感染SARS-CoV-2
<ol><li>培养维罗（猴子肾上皮细胞）细胞在杜尔贝科的修改鹰介质含有5%的胎儿牛血清。<ol>
<li>种子 50，000 细胞到盖片 （No. 1， 15 × 15 毫米） 在 12 井板.</li>
	</ol>
</li>
	<li>在透气跨井插入支架上用 膜（直径 = 6.5 mm） 和支气管上皮生长介质培养培养物，以备不时之用。</li>
	<li>病毒感染<ol>
<li>接种SARS-CoV-2的细胞，每mL组织培养中位数感染剂量（TCID50）</li>
		<li>在37°C下孵化细胞2小时。</li>
		<li>用 PBS 清洗两次细胞以去除未绑定病毒。</li>
		<li>培养细胞为48小时。</li>
	</ol>
</li>
</ol>4. 在盖片培养的维罗细胞中的 SARS-CoV-2 RNA-FISH
第 1 天
<ol><li>修复和渗透细胞<ol>
<li>在 RT 使用 3.7% 的 w/v PFA 解决方案修复受感染的细胞，使用 1 小时。</li>
		<li>吸气 3.7% PFA 溶液，使用 1 倍 PBS 洗涤细胞两次。</li>
		<li>在 RT 中用 PBST 溶液使细胞渗透 10 分钟，并激动地进行。</li>
		<li>吸气PBST，用1倍PBS洗两次细胞。</li>
	</ol></li>
	<li>检波<ol>
<li>吸气1倍PBS溶液，并在RT用2x SSC洗两次细胞。</li>
		<li>吸气 2x SSC 溶液，并通过添加至少 300 μL 的探针杂交缓冲来预混合样品。盖住含有细胞的井，在37°C孵育30分钟。</li>
		<li>通过在探头混合缓冲区中加入 1.2 pmol 的探针混合物来准备探针解决方案。<ol>
<li>使用 1 μM 探头库存的 1.2 μL 来准备 300 μL 的工作库存。</li>
		</ol>
</li>
		<li>取出预贿赂溶液，并将盖片转移到加湿室。<ol>
<li>将探针溶液的 30-50μL 吹到胶片上，形成单个液滴。</li>
		</ol>
</li>
		<li>在 37 °C 处过夜（12-18 h） 孵化样品。 第2天</li>
		<li>将盖片重新转移到 12 井板中，并在 37 °C 下用 400 μL 的探头清洗缓冲器清洗 4 x 5 分钟，去除多余的探头溶液。 注意:作为将 30-50μl 别名放在底片上和覆盖膜下进行孵化的替代方案，请直接添加 300 μL 的探针溶液，以覆盖 12 井板中的盖片。此过程更简单，但需要大量的试剂。使用前将探头洗涤缓冲器加热至 37 °C。计算所需的缓冲区量，并将其转移到 15 mL 锥形离心机管中。</li>
		<li>在RT使用5x SSCT清洗样品2 x 5分钟。</li>
		<li>将 5 倍 SSCT 解决方案替换为 1 倍 PBS，并将样品存储在 4 °C，直到放大。</li>
	</ol></li>
	<li>放大<ol>
<li>从井中取出 1 倍 PBS 溶液，并在每个井中加入至少 300 μL 的放大缓冲。在 RT 孵化样品 30 分钟。</li>
		<li>通过在单独的管中快速冷却所需的体积来准备每个HCR发夹（h1和h2）。<ol>
<li>要准备 300 μL 的放大溶液，请使用每个发夹的 18 pmol（例如，对于 300 μL，使用 3 μM 库存发夹溶液的 6 μL）。</li>
			<li>将发夹溶液转移到管中。</li>
			<li>在95°C孵育90年代。</li>
			<li>在黑暗中冷却到RT30分钟。</li>
		</ol>
</li>
		<li>通过在放大缓冲区中添加快速冷却的"h1"和"h2"发夹来准备发夹混合物。</li>
		<li>将30-50μL的发夹混合物滴入胶片上。</li>
		<li>在 RT 的黑暗中在夜间 （12-18 h） 孵化样品。 第3天</li>
		<li>将盖片移回 12 个井板，并通过在 RT 中搅拌 5 x 5 分钟，用 5 倍 SSCT 去除多余的发夹。</li>
		<li>吸气 5 倍 SSCT 缓冲区，代之以 1 倍 PBS 。 注意:如有必要，请在标准免疫荧光检测中使用RNA-FISH样本，然后对核进行染色（见第5节）。</li>
	</ol></li>
	<li>核染色和滑动安装<ol>
<li>吸气 1x PBS 解决方案，并在 1x PBS 解决方案中将其替换为 4+，6-二恶英-2-苯林多（DAPI，0.2μg/mL）。</li>
		<li>在黑暗中在RT孵化样品10分钟。</li>
		<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
		<li>放置两滴10μL的安装介质:确保将滴子充分分离，以便将两个盖片放置在单个幻灯片上。</li>
		<li>通过敲击干净毛巾上的盖子，去除多余的液体，然后将它们置于反法德安装介质中，细胞朝下。</li>
		<li>将安装的样品放在干燥平坦的黑暗表面，让它们进行固化。</li>
		<li>固化后，用 VALAP 密封剂或指甲油密封盖片的边缘，以防止样品干燥。</li>
	</ol></li>
</ol>5. HAE 文化中的 SARS-CoV-2 RNA-鱼
第 1 天
<ol start="1"><li>修复和渗透 HAE 文化<ol>
<li>吸气介质，并在RT使用3.7%PFA溶液修复受感染的细胞1小时。</li>
		<li>吸气 3.7% PFA 溶液，用 1 倍 PBS 洗两次细胞。<ol>
<li>在跨井插入下，用 1 倍 PBS 替换 3.7% 的 PFA 解决方案。</li>
		</ol>
</li>
		<li>丢弃 PBS，然后使用 2 x 5 分钟洗涤器使样品脱水，100% MeOH 预制为 -20 °C。</li>
		<li>第二次洗涤后，用新鲜的冷MeOH代替缓冲区，在Transwell插入下渗透。在-20°C过夜。</li>
	</ol>
</li>
</ol>第2天
<ol start="2"><li>再水化 通过冰上分级的 MeOH/PBST 解决方案系列（每组 5 分钟）补充样品水分:75% MeOH/25% PBST、50% MeOH/50% PBST、25% MEOH/75% PBTS 和 100% PBST（两次）。<ol>
<li>在冰上用 50% 5 倍 SSCT/50% PBST 清洗细胞 5 分钟。</li>
		<li>用5倍SSCT在冰上清洗细胞5分钟。</li>
		<li>用 1 倍 PBS 替换 5 倍 SSCT 缓冲区。</li>
	</ol>
</li>
	<li>检波<ol>
<li>在冰上用100μL的探针杂交缓冲器孵化细胞（在Transwell插入器内）5分钟。接下来，在37°C（预灌木化）下将盘子转移到孵化器30分钟。 注意:探头混合缓冲区在使用前必须预热至 37 °C。计算所需的音量:单个跨井插入需要 100 μL。</li>
		<li>准备探头解决方案。由于 1 mL 的探头解决方案需要 4 pmol 的探针，因此在 1 mL 的探针杂交缓冲区中加入 1 μL 的 1 μM 探头库存解决方案，并很好地混合。 注意:对于RNA检测，请使用每个跨井插入100μL的探针溶液。将探针溶液留在冰上，直到预贿赂步骤结束。</li>
		<li>删除预贿赂解决方案，并添加探头解决方案。</li>
		<li>在 37 °C 处隔夜（12-18 h） 孵化细胞。 第3天</li>
		<li>通过在 37 °C 下使用 100 μL 的探头清洗缓冲器清洗 4 x 15 分钟来去除多余的探头。</li>
		<li>在RT使用5x SSCT清洗样品2 x 5分钟。</li>
		<li>将 5 倍 SSCT 替换为 1 倍 PBS，并将样品存储在 4 °C，直到放大。</li>
	</ol>
</li>
	<li>放大<ol>
<li>通过在 RT 上用放大缓冲器孵化样品 30 分钟来预加样品。</li>
		<li>通过在单独的管中快速冷却所需的体积来准备每个发夹。<ol>
<li>要准备 500 μL 的放大溶液，请使用每个发夹的 30 pmol（例如，对于 500 μL，使用 10 μL 的 3 μM 库存发夹溶液）。</li>
			<li>将发夹溶液转移到管子上。</li>
			<li>在95°C孵育90年代。</li>
			<li>在黑暗中冷却到RT30分钟。</li>
		</ol>
</li>
		<li>通过在 RT 将所有扣冷发夹添加到 500 μL 的放大缓冲器中，准备发夹解决方案。</li>
		<li>删除预加放大解决方案，并添加完整的发夹解决方案。</li>
		<li>在黑暗中在 RT 过夜 （12-18 h） 孵化样品。</li>
		<li>通过在 RT 中用 5 倍 SSCT 清洗去除多余的发夹，具体如下: 2 x 5 分钟、2 x 15 分钟和 1 x 5 分钟。</li>
		<li>将 5 倍 SSCT 解决方案替换为 1 倍 PBS，并在 4 °C 中存储不超过 2-3 天，或直接进行核染色。 注意:如有必要，请在标准免疫荧光检测中使用RNA-FISH样本，然后进行核染色（见第6节）。</li>
	</ol>
</li>
	<li>核染色和滑动安装<ol>
<li>吸气 1x PBS 解决方案，并在 1x PBS 中将其替换为 DAPI 解决方案 （0.2μg/mL）。</li>
		<li>在黑暗中在RT孵化样品10分钟。</li>
		<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
		<li>将 Transwell 插入的切口膜放置在 10 μL 的抗法德安装介质上，将细胞朝上，并在膜上添加额外的安装介质 （5 μL）。</li>
		<li>用盖子盖住膜。</li>
		<li>在黑暗中将安装的样品涂在干燥平坦的表面上。</li>
		<li>固化后，用 VALAP 密封剂或指甲油密封盖片的边缘，以防止样品干燥。</li>
	</ol>
</li>
</ol>6. 维罗细胞和HAE培养物的免疫荧光分析
注意:在第3天对细胞系进行免疫荧光检测，或对HAE培养物进行第4天免疫荧光检测。对每个模型使用不同的方法。所有差异都清楚表明。
<ol><li>从井中吸气 1 倍 PBS 解决方案。</li>
	<li>在 PBST 溶液中用 1% w/v 牛血清白蛋白在 37 °C 下孵化 30 分钟来阻止样品。</li>
	<li>通过在阻断溶液中准备适当的稀释并孵育，准备主要抗体。<ol>
<li>对于盖片上的维罗细胞:<ol>
<li>将30-50μL的抗体溶液滴入湿度室的胶片上。</li>
			<li>将盖子盖在抗体滴上，细胞朝下。</li>
		</ol>
</li>
		<li>对于 HAE，用抗体溶液 （100 μL） 替换插入物中的阻塞剂，并将样品孵化在加湿室中。 注意:用原抗体调整每次孵化的时间和温度。典型的参数是2小时在RT或过夜在4°C。</li>
	</ol>
</li>
	<li>使用PBST清洗样品3 x 5分钟。<ol>
<li>对于盖片上的细胞，转移到12井板，并添加PBST解决方案。</li>
		<li>对于 HAE 文化，用 PBST 解决方案替换抗体解决方案。</li>
	</ol>
</li>
	<li>检查可用于对焦显微镜的光源和过滤器。</li>
	<li>根据宿主物种选择次生抗体。根据可用光源选择荧光参数（激发和发射波长、光谱宽度和激发效率）。 注:光谱参数可以使用在线工具进行建模（参见 材料表）。</li>
	<li>通过用阻塞溶液稀释适当的二次抗体解决方案，准备适当的二次抗体解决方案。</li>
	<li>用二次抗体孵育（如在6.3），但在37°C孵育1小时。</li>
	<li>将样品按步骤 6.4 清洗。</li>
	<li>核染色和滑动安装<ol>
<li>用于盖片上的单元格<ol>
<li>吸气PBST，并在1x PBS解决方案中将其替换为DAPI（0.2微克/mL）。</li>
			<li>在黑暗中在RT孵化样品10分钟。</li>
			<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
			<li>将盖片放在 10 μL 的安装介质滴上，细胞朝下。</li>
			<li>用指甲油密封盖子。</li>
		</ol>
</li>
		<li>对于HAE文化<ol>
<li>吸气1倍PBST，并在1x PBS溶液中替换为DAPI（0.2μg/mL）。</li>
			<li>在黑暗中在RT孵化样品10分钟。</li>
			<li>吸气 DAPI 溶液，用 1 倍 PBS 洗两次细胞。</li>
			<li>将切出的膜放在 10 μL 的安装介质滴上，将细胞朝上，并在膜上添加额外的 （5 μL） 安装介质。</li>
			<li>用盖子盖住膜。</li>
			<li>用指甲油密封盖子。</li>
		</ol>
</li>
	</ol>
</li>
</ol>7. 聚焦显微镜
<ol><li>通过指定使用的氟磷来定义轨道。</li>
	<li>选择扫描模式和速度。</li>
	<li>通过将每个氟磷与各自的负控制进行比较来调整每个荧光素的激光功率、增益和偏移值:用于病毒、模拟感染细胞:对于细胞蛋白，样品沾染了来自适当宿主的异型控制抗体。</li>
	<li>要获取 3D 图像，请激活 z 堆栈模式，并设置顶部和底部限制。设置步数/数字。 注:有关冠状病毒成像的更多详细信息，请参阅19。</li>
</ol>

<ol>
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作者没有利益冲突可以申报。

免疫-RNA-FISH是RNA和细胞蛋白双重染色的可靠方法。在这里，描述了一个经过修改的免疫-RNA-FISH协议，允许检测SARS-CoV-2RNA和细胞蛋白的细胞系和HAE培养物。此协议可适用于不同的细胞模型，包括细胞单层或特定组织。该方法依赖于由适当的探头本地化启动的HCR的概念。接下来，使用分发启动器探头开始放大荧光标记放大器的信号，当使用共聚焦显微镜观察时，可产生极小到无背景荧光。放大器可以...

SARS-CoV-2是一种新型的人类β可罗纳病毒，于2019年底出现，几个月后引发了前所未有的大流行。由于该病毒是科学的新生病毒，因此其大部分生物学及其对宿主细胞的影响仍不得而知。因此，如果要了解病毒细胞和组织在感染过程中的三胞头，其基本的生物学特征及其对宿主的影响就很重要。使用多种技术来检查病毒宿主之间的相互作用，包括生化、生物和物理检测。原位杂交是一种常用方法，它采用标记的互补DNA、RNA或改性核酸探针，该探针定位到细胞或组织中的特定DNA或RNA序列。
一种新的RNA荧光原位杂交（RNA-FISH）方法已经开发出来，它结合了修改，通过HCR1放大信号与噪声比来增加灵敏度。HCR 允许在单细胞级别上研究 RNA 本地化。由于其特异性、灵敏度和分辨率高，这种方法不仅对基础科学研究有用，而且对应用项目（如诊断）也很有用。最近，该方法的可行性被证明用于检测SARS-CoV-2RNA本地化为完全分化的3D人类气道上皮（HAE）培养物2中的附属细胞。HAE文化是用于研究"自然感染"微环境3、4背景下病毒感染的最先进工具之一。
包括SARS-CoV-2在内的几份关于人类冠状病毒（HCoV）的报告强调了表观遗传学对HCoV感染和病理生理学（在5中回顾）的重要性，例如编码血管紧张素转换酶2（ACE-2）受体6、7的基因的甲基化模式。有趣的是，质谱筛查发现了几个与SARS-CoV-2蛋白体8相互作用的表观遗传因素。更具体地说，非结构蛋白 5 （NSP5） 与表观遗传调节器、高石脱乙酰酶 2 结合，催化不活跃 NSP5 （C145A） 与 tRNA 甲基转移酶 1 （24） 相互作用。此外，NSP16甲基转移酶活性被甲基转移酶抑制剂西内芬金9阻断。然而，这些表观遗传因素在COVID-19中的确切作用尚不清楚。HCoV的复制发生在受感染细胞的细胞质中，并触发由表观遗传修饰调节的炎症反应10。
例如，HCoV-229E微调核因子-卡帕B信号，并通过在某些区域增加H3K36和H4K5的乙酰化，对宿主细胞色度图进行深刻重新编程。中东呼吸系统综合症相关冠状病毒感染增加了H3K27me3的水平，并耗尽了H3K4me3在特定干扰素敏感基因12子集的促进区域。此外，病毒RNA触发细胞免疫反应，如病毒13，逆转录病毒14，15和冠状病毒16证明。病毒RNA上的表观遗传标记可能起到细胞传感器识别的作用，如人类免疫缺陷病毒-1 RNA17的m7A甲基化所示。然而，问题依然存在:SARS-CoV-2RNA对免疫反应有何影响，是否涉及表观遗传标记？
在这里，介绍了一种优化的RNA-FISH方法，以及细胞系和3D组织（完全分化HAE）的免疫荧光分析。虽然细胞学方法，如FISH和免疫荧光，被广泛使用，这种基于HCR的新一代原位杂交方法从未用于病毒检测（除了在最近的出版物）2。一般来说，免疫染色和鱼氧化物需要以下步骤:渗透，使探针或抗体穿透:固定其中细胞材料固定和保存;检测其中应用抗体或核酸探针;最后，安装样品进行可视化。
虽然现有协议具有这些通用功能，但它们在所涉及的参数方面存在显著差异。在这里，一个优化的，简单的，免疫-RNA-FISH协议已经描述检测SARS-CoV-2RNA在HAE培养和维罗细胞。该技术包括以下步骤:（1） 用甲醛固定细胞:（2） 用洗涤剂或甲醇（MeOH） 渗透:（3） 通过一系列分级的 MeOH 解决方案（仅限 HAE 文化）进行补液:（4） 检测:（五）利用HCR技术检测SARS-CoV-2RNA的放大:（六）免疫学:和（7）在共焦显微镜下成像。

<table><tbody><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Confocal Microscope LSM 880</td><td>ZEISS </td><td></td><td></td></tr><tr><td>Grant Bio, Mini Rocker- Shaker</td><td>Fisher Scientific</td><td>12965501</td><td></td></tr><tr><td>Incubator Galaxy170R</td><td>New Brunswick</td><td>CO170R-230-1000</td><td></td></tr><tr><td>Thermomixer Comfort</td><td>Eppendorf</td><td>5355 000 011</td><td></td></tr><tr><td>Materials</td><td></td><td></td><td></td></tr><tr><td>15 mm x 15 mm NO. 1 coverslips</td><td>LabSolute</td><td>7695022</td><td></td></tr><tr><td>1.5 mL tubes</td><td>FL-MEDICAL</td><td>5.350.023.053</td><td></td></tr><tr><td>12-well plate</td><td>TTP</td><td>92412</td><td></td></tr><tr><td>Conical centrifuge tube</td><td>Sarstedt</td><td>5.332.547.254</td><td></td></tr><tr><td>parafilm</td><td>Sigma</td><td>P7793-1EA</td><td></td></tr><tr><td>serological pipets</td><td>VWR Collection</td><td>612-5523P, 612-5827P</td><td></td></tr><tr><td>slide glass</td><td>PTH CHEMLAND</td><td>04-296.202.03</td><td></td></tr><tr><td>Transwell ThinCerts</td><td>Grainer bio-one</td><td>665641</td><td></td></tr><tr><td>Reagents</td><td></td><td></td><td></td></tr><tr><td>Alexa fluorophore 488-conjugated secondary antibodies </td><td>Invitrogen</td><td></td><td></td></tr><tr><td>&amp;beta;5-tubulin </td><td>Santa Cruz Biotechnology</td><td> sc-134234</td><td></td></tr><tr><td>DAPI</td><td>Thermo Scientific</td><td>D1306</td><td></td></tr><tr><td>Disodium phosphate</td><td>Sigma</td><td>S51136-500G</td><td></td></tr><tr><td>EGTA</td><td>BioShop</td><td>EGT101.25</td><td></td></tr><tr><td>HCR Amplification Buffer</td><td>Molecular Instruments, Inc.</td><td>BAM01522</td><td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td></tr><tr><td>HCR amplifier B1-h1 Alexa Fluor 647</td><td>Molecular Instruments, Inc.</td><td>S013922</td><td></td></tr><tr><td>HCR amplifier B1-h2 Alexa Fluor 647</td><td>Molecular Instruments, Inc.</td><td>S012522</td><td></td></tr><tr><td>HCR Probe Hybridization Buffer</td><td>Molecular Instruments, Inc.</td><td>BPH03821</td><td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td></tr><tr><td>HCR probe set for SARS-CoV-2 Ncapsid</td><td>Molecular Instruments, Inc.</td><td>PRE134</td><td></td></tr><tr><td>HCR Probe Wash Buffer</td><td>Molecular Instruments, Inc.</td><td>BPW01522</td><td>Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td></tr><tr><td>HEPES</td><td>BioShop</td><td>HEP001.100</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>63138-250G</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>32213-1L-M</td><td></td></tr><tr><td>Monopotassium phosphate</td><td>Sigma</td><td>P5655-100G</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148-1KG</td><td></td></tr><tr><td>PIPES</td><td>BioShop</td><td>PIP666.100</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P5405-250G</td><td></td></tr><tr><td>Prolong Diamond Antifade Mounting Medium</td><td>Invitrogen</td><td>P36970</td><td></td></tr><tr><td>Sodium Chloride</td><td>BioShop</td><td>SOD001.5</td><td></td></tr><tr><td>Trisodium Citrate 2-hydrate</td><td>POCH</td><td> 6132-04-3</td><td></td></tr><tr><td>Tween-20</td><td>BioShop</td><td>TWN580.500</td><td></td></tr><tr><td></td><td>Software</td><td></td><td></td></tr><tr><td>Fluorescence Spectraviewer</td><td></td><td></td><td>Modeling spectral parameters</td></tr><tr><td>ImageJ Fiji</td><td></td><td></td><td>Acquiring and processing z-stack images</td></tr></tbody></table>

visualization of sars-cov-2 using immuno rna-fluorescence in situ hybridization

本手稿提供了原位杂交链反应 （HCR） 与免疫荧光的协议，可直观地将严重急性呼吸系统综合征冠状病毒 2 （SARS-CoV-2） RNA 在细胞系中和人类气道上皮的三维 （3D） 培养物中。该方法允许高度具体和敏感的病毒RNA可视化，依靠HCR启动的探针本地化。拆分启动器探头通过荧光标记的放大器帮助放大信号，导致共焦显微镜中可忽略不计的背景荧光。用不同的荧光染料标记放大器有助于同时识别各种目标。这反过来又使组织中的感染图谱能够更好地了解单细胞水平的病毒发病机能和复制。将这种方法与免疫荧光相结合，可以促进更好地了解宿主-病毒相互作用，包括宿主表观基因组和免疫反应通路的交替。由于敏感和特定的HCR技术，此协议也可以用作诊断工具。同样重要的是要记住，该技术可能很容易修改，以便能够检测任何RNA，包括将来可能出现的非编码RNA和RNA病毒。

本手稿中描述的免疫-RNA-FISH协议使用两个蜂窝系统执行:维罗细胞系和3D HAE培养物。两个蜂窝模型的主要步骤都显示在 表 2 中。RNA-FISH 在 HAE 培养中可视化 SARS-CoV-2 的协议包括组织样本的典型步骤，即通过一系列 MeOH-PBS 和 0.1% Tween 解决方案，以 100% MeOH 渗透和补液。免疫荧光是在RNA-FISH完成之后进行的。获取和处理了 Zstack 图像。
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Watch this Scientific Journal Video about Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization at JoVE.com

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

在这里，我们描述了一个简单的方法，结合RNA荧光原位杂交（RNA-FISH）与免疫荧光可视化严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）RNA。该协议可以增加对SARS-CoV-2 RNA-主机在单细胞水平上的分子特征的理解。

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

visualization-sars-cov-2-using-immuno-rna-fluorescence-situ

Research

JoVE Journal

Immunology and Infection

6.0K 次观看.  Jagiellonian University. 在这里，我们描述了一个简单的方法，结合RNA荧光原位杂交（RNA-FISH）与免疫荧光可视化严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）RNA。该协议可以增加对SARS-CoV-2 RNA-主机在单细胞水平上的分子特征的理解。

视频: Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

<meta charset="utf8"></head><body>下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

科研

遗传学

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

生物学

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 ...

神经科学

FISH 在人骨肉瘤细胞中长链非编码 RNA 定位

RNA Fluorescence In Situ Hybridization for Long Non-Coding RNA Localization in Human Osteosarcoma Cells

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition (EMT), migration, infiltration, and autophagy of cancer cells. Localization detection of lncRNAs in cells can provide insight into their functions. By designing the lncRNA-specific antisense chain sequence followed by labeling with fluorescent dyes, RNA fluorescence in situ hybridization (FISH) can be applied to detect the cellular localization of lncRNAs. Together with the development of microscopy, the RNA FISH techniques now even allow for visualization of the poorly expressed lncRNAs. This method can not only detect the localization of lncRNAs alone, but also detect the colocalization of other RNAs, DNA, or proteins by using double-color or multicolor immunofluorescence. Here, we have included the detailed experimental operation procedure and precautions of RNA FISH by using lncRNA small nucleolar RNA host gene 6 (SNHG6) in human osteosarcoma cells (143B) as an example, to provide a reference for researchers who want to perform RNA FISH experiments, especially lncRNA FISH.

作者聚焦：用于在骨肉瘤细胞中定位 lncRNA-SNHG6 的 RNA FISH 

The present protocol describes a method of RNA fluorescence in situ hybridization to localize the lncRNAs in human osteosarcoma cells.

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

The important roles of long non-coding RNAs (lncRNAs) in cancer have been studied, such as regulating the proliferation, epithelial-mesenchymal transition ...

癌症研究

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

免疫与感染

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

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.

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

摘要

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下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位

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

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

荧光检测病毒RNA

全山RNA荧光

使用免疫RNA-荧光原位杂交对SARS-CoV-2进行可视化

摘要

探索更多视频

下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

一种技术，同时可视化病毒特异性CD8 + T细胞和病毒感染的细胞原位

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

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光 原 位杂交在人骨肉瘤细胞中长链非编码RNA定位

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

荧光检测病毒RNA

全山RNA荧光

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

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位

RNA荧光原位杂交在人骨肉瘤细胞中长链非编码RNA定位