这项工作全部或部分得到了美国国立卫生研究院的支持（R01 AI121315，R01 AI146017，R01 AI148382，R01 AI120860，R37 AI076119和U54 AI150472）。我们感谢Raymond F. Schinazi博士和Sadie Amichai提供感染甲型流感病毒的细胞。

1.盖玻片或腔室载玻片上的种子细胞（悬浮液 与 贴壁细胞）（提供的方案使用盖玻片）。
<ol><li>悬浮细胞的接种<ol><li>通过首先将盖玻片在乙醇（EtOH）中孵育5分钟（灭菌盖玻片并去除任何残留物）来制备聚-D-赖氨酸（PDL）包被的盖玻片（促进悬浮细胞粘附到盖玻片上）。然后在磷酸盐缓冲盐水（PBS）中洗涤2x，然后在室温（RT）下将盖玻片在PDL（20μg/ mL）中孵育30分钟（分钟）。</li>
		<li>去除 PDL 并在 PBS 中洗涤 2 次。沉淀细胞（先前根据所需的成像条件感染/处理）并将 106 个细胞重悬于 50 μL PBS 中。在玻璃上点缀 50 μL 细胞（PDL 包衣），并在室温下孵育 30 分钟。</li>
	</ol></li>
	<li>贴壁细胞的接种<ol><li>将细胞放在无菌盖玻片上培养，置于6孔培养皿中，用病毒颗粒感染细胞或用感兴趣的化合物处理。在成像实验的样品制备之前，让细胞达到50-70%的汇合度。</li>
	</ol></li>
</ol>2. 细胞固定:保存细胞形态以进行荧光成像研究
注意:在细胞固定前，将 4% PFA 和 PBS 保持在室温至少 30 分钟。
<ol><li>吸出细胞培养基，在PBS中在盖玻片上洗涤细胞3次，并将细胞固定在4%多聚甲醛（PFA）中30分钟。吸出PFA并在PBS 2x中洗涤细胞。 注意:如果实验者希望在另一个日期恢复实验，细胞现在可以脱水和储存。固定细胞在储存前可以在EtOH中脱水。<ol><li>细胞固定后洗涤后去除PBS，并用500μL的50%EtOH（水中v / v）替换。在室温下孵育5分钟。</li>
		<li>去除 50% 的 EtOH，并用 500 μL 的 70% EtOH 代替。在室温下孵育5分钟。</li>
		<li>去除 70% 乙醇，并用 500 μL 100% 乙基醇代替。在室温下孵育5分钟。</li>
		<li>去除100%环氧乙烷，并用新鲜的100%乙醇代替。 注意:脱水细胞可以在-20°C下储存6个月。储存前用胶带或封口膜密封板，以防止乙二醇蒸发。</li>
		<li>对细胞进行再水化以转移到细胞/病毒的多重荧光标记上。</li>
		<li>去除 100% 乙醇，并用 500 μL 70% 乙基醇代替。在室温下孵育2分钟。 注意:任何时候都不要让细胞变干。始终使用足够的溶液来淹没所有细胞。</li>
		<li>去除 70% 的 EtOH，并用 500 μL 的 50% EtOH 代替。在室温下孵育2分钟。</li>
		<li>去除 50% 的 EtOH，并用 1x PBS 代替。 注意:在蛋白酶处理（步骤4） 和目标探针应用（步骤5）之前准备蛋白酶稀释液，杂交探针和洗涤缓冲液。有关试剂制备的具体说明在每个相应步骤的开头提供。</li>
	</ol></li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>其他注意事项</td>
		</tr><tr><td>蛋白酶溶液</td>
			<td>在 1X PBS 中制备</td>
		</tr><tr><td>目标寡核探针</td>
			<td>在杂交缓冲液中稀释</td>
		</tr><tr><td>洗涤缓冲液</td>
			<td>按照靶标杂交步骤洗涤</td>
		</tr><tr><td>杂交缓冲液</td>
			<td>配方如表3所示</td>
		</tr></tbody></table>表 1:MICDDRP 方案中的关键试剂。
3. 细胞透化:增加大分子（抗体、核酸杂交探针）进入细胞和细胞器的通道
<ol><li>细胞固定或补液后取出 1x PBS，并在 1x PBS 中用 500 μL 的 0.1% （v/v） 吐温-20 替换。在室温下孵育10分钟。逐个替换。用 500 μL 1x PBS 洗涤一次，然后加入新鲜的 1x PBS。</li>
</ol>4. 玻片盖 玻片 固定和 蛋白酶处理 ，去除固定病毒/细胞核酸中的核酸结合蛋白，提高杂交效率
注意:在细胞透化过程中制备稀释的蛋白酶III（参见 材料表中的试剂细节）溶液。让蛋白酶在透化前达到室温10分钟。
<ol><li>将一小滴指甲油放在无菌载玻片上。干燥背部（没有细胞层的一面），并将盖玻片的边缘放在指甲油滴上，粘附细胞朝上的一侧朝上。在固定的盖玻片上加入几滴PBS以防止干燥。<ol><li>使用疏水屏障笔（使试剂定位在细胞上的憎水笔）在现在粘附在载玻片上的盖玻片周围画一个圆圈（距离盖玻片约 3 毫米）。</li>
	</ol></li>
	<li>在 1x PBS（100 μL/盖玻片）中稀释蛋白酶 III。 注意:蛋白酶浓度可能需要根据细胞类型、探针或靶核酸的差异通过经验优化进行调整（参见 讨论）。为了在不同病毒系统中实现最有效的RNA / DNA标记，我们成功地进行了下表2所示的以下稀释，稀释范围为（1至 2 ）-（1至15）（蛋白酶至1x PBS）。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>探测目标</td>
			<td>蛋白酶稀释在 1X PBS 中（蛋白酶 III 至 1X PBS）</td>
		</tr><tr><td>HIV-1 DNA/RNA</td>
			<td>1 到 5</td>
		</tr><tr><td>HTLV-1 脱氧核糖核酸/核糖核酸</td>
			<td>1 到 5</td>
		</tr><tr><td>乙肝病毒核糖核酸和总乙肝病毒核糖核酸</td>
			<td>1 到 15</td>
		</tr><tr><td>亚病毒核酸核糖核酸</td>
			<td>1 到 15</td>
		</tr><tr><td>齐克病毒核糖核酸</td>
			<td>1 至 2</td>
		</tr></tbody></table>表2:用于病毒核酸杂交的PBS中的蛋白酶III稀释液。
<ol start="3"><li>固定后在盖玻片上倒出1x PBS，并应用稀释的蛋白酶III。</li>
	<li>在40°C的加湿烤箱中孵育15分钟。</li>
	<li>倒出蛋白酶溶液并将载玻片浸没在 1x PBS 中。在室温下用摇摆盘搅拌2分钟，用新的1x PBS重复洗涤。<ol><li>对于仅DNA检测，用无核酸酶水洗涤样品三次，每次2分钟，然后用在PBS中稀释的5mg / mL RNase A在37°C下孵育30分钟。</li>
		<li>倒出RNase A溶液，用超纯水洗涤3次2分钟。继续对目标探针进行 ISH。 注意:杂交缓冲液可在不影响所提供结果的vRNA染色效率的情况下改进vDNA检测（代表性结果）。用杂交探针 1:1 稀释 DNA 通道 1 （C1） 探针。在杂交缓冲液中稀释RNA C2和C3探针。我们的成像研究中使用的C2和C3探针采用50X溶液（1:50;靶探针 到 杂交缓冲液）。</li>
	</ol></li>
	<li>按照以下分步程序在无核酸酶水中制备杂交缓冲液:<ol><li>在 15 mL 管中，加入 700 μL 无核酸酶水、300 μL 50%（重量/体积 （w/v））葡聚糖硫酸盐、300 μL 5 M NaCl、125 μL 200 mM 柠檬酸钠 （pH 6.2） 和 375 mg（粉末）碳酸乙烯酯。</li>
		<li>使用涡旋充分混合以溶解碳酸乙烯酯（确保所有粉末溶解并澄清溶液）。</li>
		<li>加入 25 μL 10%（体积/体积 （v/v））吐温-20 和足够的无核酸酶水以完成 2.5 mL（2x 溶液）。 注意:所示杂交缓冲液的配方可在下面的 表3 中找到。溶液稳定一周。确保吐温-20洗涤剂充分混合，同时注意防止溶液起泡。</li>
	</ol></li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>试剂</td>
			<td>库存集中度</td>
			<td>附加说明</td>
		</tr><tr><td>无核酸酶水</td>
			<td>那</td>
			<td></td>
		</tr><tr><td>葡聚糖硫酸盐</td>
			<td>50% （w/v）</td>
			<td>非常粘稠</td>
		</tr><tr><td>氯化钠</td>
			<td>5 米</td>
			<td></td>
		</tr><tr><td>柠檬酸钠（pH 6.2）</td>
			<td>200 毫米</td>
			<td>储存在4°C</td>
		</tr><tr><td>碳酸乙烯酯</td>
			<td>那</td>
			<td>粉</td>
		</tr><tr><td>吐温-20</td>
			<td>10% （V/V）</td>
			<td></td>
		</tr></tbody></table>表3:试剂杂交缓冲液列表。
5. 与 DNA/RNA 靶标杂交探针孵育:靶寡核苷酸探针与目标区域结合，形成用于前置放大器、放大器和荧光探针结合的分子支架。
注意:如果没有包括RNase处理，则在40°C下加热DNA / RNA探针10分钟（在细胞透化期间），并冷却至RT至少10分钟。如果在靶标探针杂交之前需要RNase处理或进一步的样品处理，请相应地加热探针。升温后旋转C2和C3探针，并在杂交缓冲液中稀释。稀释后，可以短暂加热C2和C3探头。在40°C下温热50x洗涤缓冲液（详见 材料表 ）10-20分钟，并在分子生物学级水中稀释至1x。
<ol><li>在加入杂交探针之前，将 200 μL 杂交缓冲液在 67 °C 下孵育 10 分钟。在杂交缓冲液中稀释探针（ 配方如表2所列）。加入 50 μL/盖玻片。</li>
	<li>在40°C的加湿炉中孵育2小时（h）。倾析探针并将载玻片浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿搅拌2分钟，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>6. 扩音器（放大器）1-FL 杂交:添加与靶 DNA/RNA 探针尾序列互补的前置扩音器（步骤 5）
注意:放大器在使用前应处于室温状态。使用前 30 分钟将每个单独的放大器从冰箱中取出，然后放在室温工作台上。
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 1-FL。在40°C的加湿炉中孵育30分钟。</li>
	<li>倒出放大器 1-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿2分钟搅拌，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>7. Amp 2-FL 杂交:与具有同源识别序列的信号放大器一起孵育到前置放大器 （Amp 1-FL）
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 2-FL。在40°C的加湿炉中孵育15分钟。</li>
	<li>倒出安培 2-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿2分钟搅拌，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>8. Amp 3-FL 杂交:与第二个信号放大器一起孵育
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 3-FL。在40°C的加湿炉中孵育30分钟。</li>
	<li>倾析放大器 3-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿2分钟搅拌，用新的1x洗涤缓冲液重复洗涤。</li>
</ol>9. Amp 4-FL杂交:荧光标记和最终杂交步骤
注意:首先，请参阅表 4，根据目标探头的通道选择合适的 Amp 4（A、B 或 C）- FL。评估标记目标 DNA/RNA 所需的 Amp 4-FL。下表提供了一个示例:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td>
			<td>一个</td>
			<td>B</td>
			<td>C</td>
		</tr><tr><td>DNA 通道 1 （C1）</td>
			<td>488</td>
			<td>550</td>
			<td>550</td>
		</tr><tr><td>DNA/RNA 通道 2 （C2）</td>
			<td>550</td>
			<td>488</td>
			<td>647</td>
		</tr><tr><td>核糖核酸通道 3 （C3）</td>
			<td>647</td>
			<td>647</td>
			<td>488</td>
		</tr></tbody></table>表 4:用于多重 ISH 的 Amp 4（A、B 或 C）-FL 荧光探针的选择。
注意:对于多路复用 FISH（多个靶标），选择正确的 Amp 4（A、B 或 C）- FL 对于正确标记感兴趣的靶标至关重要。目标探针（步骤5）具有不同的颜色通道（C1、C2或C3），这些通道根据所选的Amp 4-FL决定其各自的荧光标记（Alexa 488，Atto 550或Alexa 647）。选择荧光团组合进行多重成像的示例如图 2 和 图3所示。作为另一个示例，选择 Amp 4B-FL 将选择性地使用 Atto 550 标记 DNA C1 探针，使用 Alexa 647 标记 RNA C3 探针。
<ol><li>从 1x 洗涤缓冲液中取出载玻片并轻拍/吸收以去除多余的液体。</li>
	<li>在盖玻片上加入 1 滴 Amp 4-FL。在40°C的加湿炉中孵育15分钟。 注意:按照步骤9.2，盖住样品，避光。</li>
	<li>倾析放大器 4-FL 并浸没在 1x 洗涤缓冲液中。在室温下摇动培养皿搅拌2分钟，用新的1x洗涤缓冲液重复洗涤。用 1x PBS（2 分钟）清洗并存放在 PBS 中。</li>
</ol>10. 蛋白质免疫染色:标记感兴趣的蛋白质
<ol><li>倒出 PBS 并向盖玻片中加入 200 μL 封闭缓冲液（1% w/v BSA，10% v/v FBS 在 PBS 中，0.1% v/v 吐温-20 （PBST））。在室温下孵育1小时。</li>
	<li>倒出封闭缓冲液并应用在PBST + 1% w/v BSA中稀释的200 μL一抗。在室温下孵育1小时。</li>
	<li>用PBST在室温下摇动洗涤载玻片两次10分钟。</li>
	<li>在室温下以PBST + 1% w / v BSA应用所选的二抗1小时。</li>
	<li>用PBST在室温下摇动洗涤载玻片10分钟。</li>
</ol>11. 核染色:免疫染色后的复染细胞核
<ol><li>倒出PBST并在室温下应用DAPI或所选的核染色剂1分钟。</li>
	<li>用PBS在室温下摇动洗涤载玻片两次10分钟。</li>
</ol>12. 安装
<ol><li>将 1 滴抗淬灭溶液（例如 Prolong Gold）放在新的无菌载玻片上（首先，用 EtOH 清洁载玻片并晾干以确保玻璃上没有残留物）。使用相同的尖端，铺开抗淬灭溶液滴剂以覆盖大约盖玻片大小的区域。 注意:防淬灭溶液非常粘稠，可能难以移液。在移液前切断 200 μL 吸头可能会缓解这些问题。</li>
	<li>从载玻片上取下带有细胞样品的盖玻片，然后浸入PBS中，以使用镊子和PBS去除背部残留的指甲油。使用Kimwipe擦干镊子和盖玻片的背面。</li>
	<li>将盖玻片轻轻嵌入抗淬灭溶液液滴中，将盖玻片的样品侧（带有细胞层的一侧）放在液滴上）。</li>
	<li>让样品干燥过夜。</li>
</ol>13. 成像
<ol><li>用落射荧光显微镜成像。</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-copy+gene+detection+using+branched+DNA+(bDNA)+in+situ+hybridization.">Single-copy gene detection using branched DNA (bDNA) in situ hybridization.</a> Journal of Histochemistry and Cytochemistry. 49 (5), 603-612 (2001).</li><li>Battich, N., Stoeger, T., Pelkmans, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+transcriptomics+in+thousands+of+single+human+cells+at+single-molecule+resolution.">Image-based transcriptomics in thousands of single human cells at single-molecule resolution.</a> Nature Methods. 10 (11), 1127-1133 (2013).</li><li>Bushnell, 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=ProbeDesigner:+for+the+design+of+probesets+for+branched+DNA+(bDNA)+signal+amplification+assays.">ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.</a> Bioinformatics. 15 (5), 348-355 (1999).</li><li>Stultz, R. 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C., 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=HIV-1+replication+complexes+accumulate+in+nuclear+speckles+and+integrate+into+speckle-associated+genomic+domains.">HIV-1 replication complexes accumulate in nuclear speckles and integrate into speckle-associated genomic domains.</a> Nature Communications. 11 (1), 3505 (2020).</li><li>Dharan, A., Bachmann, N., Talley, S., Zwikelmaier, V., Campbell, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+pore+blockade+reveals+that+HIV-1+completes+reverse+transcription+and+uncoating+in+the+nucleus.">Nuclear pore blockade reveals that HIV-1 completes reverse transcription and uncoating in the nucleus.</a> Nature Microbiology. 5 (9), 1088-1095 (2020).</li></ol>

作者没有什么可透露的。

同时可视化 RNA、DNA 和蛋白质通常需要广泛的优化。两种常用的方法是5-乙炔基-2-脱氧尿苷（EdU）标记和DNA FISH。EdU标记已被应用于同时可视化病毒DNA和蛋白质，因为EdU被掺入新生DNA中，随后通过点击化学 用 含叠氮化物的荧光染料标记。因此，EdU标记可用于监测DNA病毒的天然病毒复制动力学或具有DNA模板的病毒进行复制12。然而，EdU标记的一个缺点是，在分裂细胞中，复制...

虽然数以千计的商业抗体可用于通过常规免疫染色方法特异性标记蛋白质，并且虽然融合蛋白可以使用光优化荧光标签进行工程设计以跟踪样品中的多种蛋白质1，但蛋白质的显微可视化通常与传统的DNA荧光原位杂交（FISH）不兼容2.使用基于荧光的方法同时可视化DNA、RNA和蛋白质的技术限制阻碍了对病毒复制的深入了解。在感染过程中跟踪病毒核酸和蛋白质使病毒学家能够可视化病毒复制和组装的基本过程3，4，5，6。
我们开发了一种成像方法，即基于d na，rNA和Protein的成像方法m ultiplex immunofluorescent cell（MICDDRP）3，该方法利用支链DNA（bDNA）原位技术来提高核酸检测的灵敏度7，8，9.此外，该方法利用成对探针来增强特异性。bDNA序列特异性探针使用分支前置放大器和放大器DNA产生强烈的局部信号，改进了以前的杂交方法，该方法依赖于靶向DNA9中的重复区域。临床环境中的受感染细胞通常不含丰富的病毒遗传物质，为诊断环境中荧光核酸检测的灵敏方法提供了商品。通过RNAscope7和ViewRNA10等方法实现bDNA技术的商业化填补了这一空白。bDNA荧光成像的灵敏度在细胞生物学中也具有重要的用途，可以检测细胞培养模型中稀缺的核酸种类。灵敏度的巨大提高使基于bDNA的成像方法适用于研究病毒。然而，一个潜在的缺点是这些方法专注于可视化RNA或RNA和蛋白质。所有复制细胞和许多病毒在其复制周期中都具有DNA基因组或形成DNA，因此非常需要能够对RNA和DNA以及蛋白质进行成像的方法。
在MICDDRP协议中，我们使用RNAscope方法进行bDNA FISH以检测病毒核酸，并修饰7。该方案的主要修改之一是化学固定后蛋白酶处理的优化。蛋白酶处理有助于去除与核酸结合的蛋白质，从而提高探针杂交效率。蛋白酶处理后用支链寡核苷酸探针孵育。应用bDNA探针后，洗涤样品，随后与信号前置放大器和放大器DNA一起孵育。多重原位杂交（ISH），标记多个基因靶标，需要具有不同颜色通道的靶标探针进行光谱分化7。用DNA扩音器孵育后进行免疫荧光（IF）。
bDNA ISH通过放大靶标特异性信号来改善信噪比，并减少非特异性杂交事件的背景噪声7，11。靶探针是使用公开可用的软件程序设计的，这些软件程序预测非特异性杂交事件的概率，并计算探针-目标杂交7，11的熔解温度（Tm）。靶探针包含一个与靶DNA/RNA序列互补的18至25碱基区域、一个间隔序列和一个14个碱基的尾部序列。一对靶探针，每个探针具有不同的尾序列，杂交到目标区域（跨越~50个碱基）。两个尾序列形成前置放大器探针的杂交位点，其中包含20个放大器探针的结合位点，此外还包含20个标记探针的结合位点。例如，核酸分子上的一千碱基（kb）区域被20个探针对靶向，从而创建一个分子支架，用于与前置放大器、放大器和标记探针进行顺序杂交。因此，这可以使每个核酸分子的理论产量达到8000个荧光标记，从而能够检测单个分子，并且比传统的FISH方法7有了巨大的改进（bDNA信号扩增示意图见图1A）。要为多路复用 ISH 设置探针，每个目标探针必须位于不同的颜色通道（C1、C2 或 C3）中。这些具有不同颜色通道的目标探针具有不同的14碱基尾序列。这些尾部序列会将不同的信号放大器与不同的荧光探针结合，从而实现跨多个目标的光谱分化。在所提出的方案中，步骤9中的表4提供了有关荧光标记靶探针的更多信息。此外，图2和图3提供了我们如何选择合适的放大器4-FL（A，B或C）（荧光探针和最终杂交步骤）的示例，以实现HIV-1和HTLV-1感染后多个病毒核酸靶标的特异性荧光标记。
我们已经展示了同时对RNA，DNA和蛋白质进行荧光可视化的几种应用，以高时空分辨率3，4，5观察病毒复制的关键阶段。例如，病毒RNA，细胞质和核DNA以及蛋白质的同时单细胞可视化使我们能够可视化HIV-1感染期间的关键事件，包括在核进入和前病毒DNA3整合之前跟踪细胞质中含有核心的RNA。此外，我们还应用MICDDRP来表征宿主因子和药物治疗对病毒感染和复制的影响4，5。在Ukah等人中，我们跟踪了用不同潜伏逆转剂处理的潜伏期细胞模型中HIV-1转录的再激活，以可视化HIV转录和潜伏期逆转4。此外，MICDDRP可以让我们可视化与小分子治疗或宿主因子限制引起的抗病毒抑制相关的表型变化。作为我们方法稳健性和广泛适用性的概念证明，我们已经证明，我们可以使用我们的方案的修改来有效地标记病毒核酸，以跟踪感染，不仅在人类免疫缺陷病毒（HIV）-1，而且在人类嗜T淋巴细胞病毒（HTLV）-1，乙型肝炎病毒（HBV），丙型肝炎病毒（HCV）， 寨卡病毒（ZIKV）和甲型流感病毒（IAV）。由于HIV-1生命周期由病毒DNA和RNA物种组成，因此我们已经根据HIV-1复制动力学对MICDDRP进行了大部分优化。然而，此外，我们已经证明，我们可以跟踪ZIKV，IAV，HBV和HCV等病毒中或两种感觉（+）和反义（-）链的不同病毒RNA转录本的合成，以监测病毒转录和复制3，4，5，6。这些研究旨在提高对几种病毒过程的机制理解，并作为将这种荧光成像技术应用于各种细胞模型的指南。

<table><tbody><tr><td>4% PFA</td><td></td><td></td><td></td></tr><tr><td>50% dextran sulfate</td><td>Amreso</td><td>198</td><td>For DNA hybridization buffer</td></tr><tr><td>50X wash buffer</td><td>ACD Bio</td><td>320058</td><td></td></tr><tr><td>6-well plates</td><td></td><td></td><td></td></tr><tr><td>Amplifier 1-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 2-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 3-FL</td><td>ACD Bio</td><td></td><td></td></tr><tr><td>Amplifier 4-FL</td><td>ACD Bio</td><td></td><td>Consult Amp-4 table in protocol</td></tr><tr><td>Anti-HCV NS5a antibody</td><td>Abcam</td><td>ab13833</td><td>Mouse monoclonal; works with HCV genotypes 1a, 1b, 3, and 4</td></tr><tr><td>Anti-HIV-1 p24 monoclonal antibody</td><td>NIH AIDS Reagent Program</td><td>3537</td><td></td></tr><tr><td>Anti-Mov10 antibody</td><td>Abcam</td><td>ab80613</td><td>Rabbit polyclonal</td></tr><tr><td>Anti-PB1 antibody</td><td>GeneTex</td><td>GTX125923</td><td>Antibody against flu protein</td></tr><tr><td>Bovine serum albumin</td><td></td><td></td><td>Blocking reagent for immunostaining</td></tr><tr><td>Cell media with supplements</td><td></td><td></td><td>Media appropriate for cell model</td></tr><tr><td>Coverslips</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>ACD Bio</td><td></td><td>Nuclear stain (RNAscope kit from ACD Bio)</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline (1X PBS)</td><td>Gibco</td><td>14190250</td><td>No calcium and magnesium</td></tr><tr><td>Ethylene carbonate</td><td>Sigma</td><td>E26258</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td></td><td></td><td>Use specific FBS based on what serum secondary antibody was raised in (e.g goat FBS)</td></tr><tr><td>Fisherbrand colorfrost plus microscope slides</td><td>Fisher Scientific</td><td>12-550-17/18/19</td><td>Precleaned</td></tr><tr><td>HCV-GT2a-sense-C2 probe</td><td>ACD Bio</td><td>441371</td><td>HCV(+) sense RNA probe</td></tr><tr><td>HIV-gagpol-C1</td><td>ACD Bio</td><td>317701</td><td>HIV-1 cDNA probe</td></tr><tr><td>HIV-nongagpol-C3</td><td>ACD Bio</td><td>317711-C</td><td>HIV-1 RNA probe</td></tr><tr><td>HybEZ hybridization oven</td><td>ACD Bio</td><td>321710/321720</td><td></td></tr><tr><td>ImmEdge hydrophobic barrier pen</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Nail polish</td><td></td><td></td><td>For immobolizing coverslip to slide prior to protease treatment</td></tr><tr><td>Nuclease free water</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>Poly-d-lysine (PDL)</td><td></td><td></td><td>Coat coverslips in 20 &amp;micro;g/mL of PDL for 30 minutes</td></tr><tr><td>Probe diluent</td><td>ACD Bio</td><td>300041</td><td>For diluting RNA C2 or C3 probes</td></tr><tr><td>Prolong gold antifade</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>Protease III</td><td>ACD Bio</td><td>322337</td><td></td></tr><tr><td>RNAscope&amp;reg;&amp;nbsp;Probe- V-Influenza-H1N1-H5N1-NP</td><td>ACD Bio</td><td>436221</td><td></td></tr><tr><td>RNase A</td><td>Qiagen</td><td></td><td></td></tr><tr><td>Secondary antibodies</td><td></td><td></td><td></td></tr><tr><td>Slides</td><td></td><td></td><td></td></tr><tr><td>Sodium chloride</td><td></td><td></td><td>For DNA hybridization buffer</td></tr><tr><td>Sodium citrate, pH 6.2</td><td></td><td></td><td>For DNA hybridization buffer</td></tr><tr><td>Tween-20</td><td></td><td></td><td>For DNA hybridization buffer and PBS-T</td></tr><tr><td>V-HBV-GTD</td><td>ACD Bio</td><td>441351</td><td>Total HBV RNA</td></tr><tr><td>V-HBV-GTD-01-C2</td><td>ACD Bio</td><td>465531-C2</td><td>HBV pgRNA probe</td></tr><tr><td>V-HCV-GT2a probe</td><td>ACD Bio</td><td>441361</td><td>HCV(-) sense RNA probe</td></tr><tr><td>V-HTLV-HBZ-sense-C3</td><td>ACD Bio</td><td>495071-C3</td><td>HTLV-1 (-) sense RNA probe targetting HBZ</td></tr><tr><td>V-HTLV1-GAG-C2</td><td>ACD Bio</td><td>495051-C2</td><td>HTLV-1 DNA probe</td></tr><tr><td>V-HTLV1-GAG-POL-sense</td><td>ACD Bio</td><td>495061</td><td>HTLV-1 (+) sense RNA probe</td></tr><tr><td>V-Influenza-H1N1-H5N1-NP</td><td>ACD Bio</td><td>436221</td><td>IAV RNA probe</td></tr><tr><td>V-ZIKA-pp-O2</td><td>ACD Bio</td><td>464531</td><td>Zika(+) sense RNA probe</td></tr><tr><td>V-ZIKA-pp-O2-sense-C2</td><td>ACD Bio</td><td>478731-C2</td><td>Zika(-) sense RNA probe</td></tr></tbody></table>

single-cell multiplexed fluorescence imaging to visualize viral nucleic acids and proteins and monitor hiv, htlv, hbv, hcv, zika virus, and influenza infection

由于缺乏能够灵敏和同时标记病毒核酸和蛋白质的强大原位杂交（ISH）技术，捕获病毒的动态复制和组装过程受到了阻碍。传统的DNA荧光原位杂交（FISH）方法通常与免疫染色不兼容。因此，我们开发了一种成像方法 MICDDRP （multiplex immunofluorescent c ell-basedd etection of D NA， RNA和 Protein），该方法能够同时进行DNA，RNA和蛋白质的单细胞可视化。与传统的DNA鱼相比，MIDDRP采用支链DNA（bDNA）ISH技术，可显著提高寡核苷酸探针的灵敏度和检测。MICDDRP的微小修饰能够同时对病毒蛋白进行成像，同时具有不同链的核酸（RNA或DNA）。我们已经应用这些协议来研究多种病毒病原体的生命周期，包括人类免疫缺陷病毒（HIV）-1，人类嗜T淋巴细胞病毒（HTLV）-1，乙型肝炎病毒（HBV），丙型肝炎病毒（HCV），寨卡病毒（ZKV）和甲型流感病毒（IAV）。我们证明了我们可以有效地标记各种病毒中的病毒核酸和蛋白质。这些研究可以为我们提供对多种病毒系统的机理理解，此外，还可以作为在广泛的细胞系统中应用DNA，RNA和蛋白质的多重荧光成像的模板。

MICDDRP的示意图如图 1所示。标记DNA和RNA之后进行免疫染色。支化放大器的使用增加了信号，允许检测单个核酸分子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61843/61843fig1v5.jpg"> 图 1:MICDDRP 和分步工作流程示意图。 （A）bDNA信号通过分支前置放大器和放大器DNA放大，以增强对病毒DNA（1）和RNA物种（...

Watch this Scientific Journal Video about Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infectio

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

这里介绍的是荧光成像方法的 协议，multiplex immunofluorescent cELL 基于d etection of DNA， RNA和 Protein（MICDDRP），该方法能够同时对不同类型和链的病毒蛋白和核酸进行荧光单细胞可视化。这种方法可以应用于各种系统。

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

single-cell-multiplexed-fluorescence-imaging-to-visualize-viral

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2.7K 次观看.  Emory University School of Medicine. 这里介绍的是荧光成像方法的 协议，multiplex immunofluorescent cELL 基于d etection of DNA， RNA和 Protein（MICDDRP），该方法能够同时对不同类型和链的病毒蛋白和核酸进行荧光单细胞可视化。这种方法可以应用于各种系统。

视频: Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

科研

免疫与感染

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

生物学

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

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 ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

使用多参数光伏平台对钙流入和HIV-1感染进行单细胞特征描述

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

<meta charset="utf8"></head><body>使用双报告基因和多重技术分析 SARS-CoV-2

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

<meta charset="utf8"></head><body>作者聚焦:通过新型免疫捕获和质谱技术推进抗病毒策略

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to understand where and when transcription is taking place within nuclear space and to visualize the relationship between episomes infected within the same cell's nucleus. Here, both immunofluorescence (IFA) and RNA-FISH have been combinedto identify actively transcribing Kaposi's sarcoma-associated herpesvirus (KSHV) episomes. By staining KSHV latency-associated nuclear antigen (LANA), it is possible to locate where viral episomes exist within the nucleus. In addition, by designing RNA-FISH probes to target the intron region of a viral gene, which is expressed only during productive infection, nascent RNA transcripts can be located. Using this combination of molecular probes, it is possible to visualize the assembly of large viral transcription factories and analyze the spatial regulation of viral gene expression during KSHV reactivation. By including anti-RNA polymerase II antibody staining, one can also visualize the association between RNA polymerase II (RNAPII) aggregation and KSHV transcription during reactivation.

Viral transcriptional factories are discrete structures that are enriched with cellular RNA polymerase II to increase viral gene transcription during reactivation. Here, a method to locate sites of actively transcribing viral chromatin in 3D nuclear space by a combination of immunofluorescence staining and in situ RNA hybridization is described.

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

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此视频中的章节

顺行运输Alphaherpes病毒传播的活细胞成像

病毒融合动力学测量方法在单粒子能级

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

荧光检测病毒RNA

使用多参数光伏平台对钙流入和HIV-1感染进行单细胞特征描述

单病毒原子力显微镜和超分辨率荧光成像的样品制备

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

基于3D 荧光显微镜的病毒转录工厂功能成像