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>
	<li>Zheng, Q., Lavis, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+photostable+fluorophores+for+molecular+imaging.">Development of photostable fluorophores for molecular imaging.</a> Current Opinion in Chemical Biology. 39, 32-38 (2017).</li><li>Marini, B., 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=Nuclear+architecture+dictates+HIV-1+integration+site+selection.">Nuclear architecture dictates HIV-1 integration site selection.</a> Nature. 521 (7551), 227-231 (2015).</li><li>Puray-Chavez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+single-cell+visualization+of+nucleic+acids+and+protein+during+HIV+infection.">Multiplex single-cell visualization of nucleic acids and protein during HIV infection.</a> Nature Communications. 8 (1), 1882 (2017).</li><li>Ukah, O. B., 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=Visualization+of+HIV-1+RNA+Transcription+from+Integrated+HIV-1+DNA+in+Reactivated+Latently+Infected+Cells.">Visualization of HIV-1 RNA Transcription from Integrated HIV-1 DNA in Reactivated Latently Infected Cells.</a> Viruses. 10 (10), (2018).</li><li>Liu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Positive+and+Negative+Sense+Viral+RNA+for+Probing+the+Mechanism+of+Direct-Acting+Antivirals+against+Hepatitis+C+Virus.">Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus.</a> Viruses. 11 (11), (2019).</li><li>Achuthan, V., 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=Capsid-CPSF6+Interaction+Licenses+Nuclear+HIV-1+Trafficking+to+Sites+of+Viral+DNA+Integration.">Capsid-CPSF6 Interaction Licenses Nuclear HIV-1 Trafficking to Sites of Viral DNA Integration.</a> Cell Host &amp; Microbe. 24 (3), 392-404 (2018).</li><li>Wang, F., 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=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14 (1), 22-29 (2012).</li><li>Xia, C., Babcock, H. P., Moffitt, J. R., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+detection+of+RNA+using+MERFISH+and+branched+DNA+amplification.">Multiplexed detection of RNA using MERFISH and branched DNA amplification.</a> Scientific Reports. 9 (1), 7721 (2019).</li><li>Player, A. N., Shen, L. P., Kenny, D., Antao, V. P., Kolberg, J. 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. D., Cenker, J. J., McDonald, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+HIV-1+Genomic+DNA+from+Entry+through+Productive+Infection.">Imaging HIV-1 Genomic DNA from Entry through Productive Infection.</a> Journal of Virology. 91 (9), (2017).</li><li>Brown, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+nuclear+proteins+together+with+transcribed+and+inactive+genes+in+structurally+preserved+cells.">Visualizing nuclear proteins together with transcribed and inactive genes in structurally preserved cells.</a> Methods. 26 (1), 10-18 (2002).</li><li>Francis, A. 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&#39;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 &#181;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&#174;&#160;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

Research

JoVE Journal

Biology

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

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

科研

生物学

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 ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

使用微量的生物规格的纳米分光光度计核酸和蛋白的浓度测定

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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 ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections.
Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile.
Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV

The role of RNA modifications in viral infections is just starting to be explored and could highlight new viral-host interaction mechanisms. In this work, we provide a pipeline to investigate m6A and m5C RNA modifications in the context of viral infections.

探索病毒感染时的m6A和m5C表继转录组:HIV示例

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as ...

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

摘要

探索更多视频

此视频中的章节