Name: temporal analysis of the nuclear-to-cytoplasmic translocation of a herpes simplex virus 1 protein by immunofluorescent confocal microscopy
Uploaded: 2018-11-04T14:00:00
Description: Watch this Scientific Journal Video about Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy at JoVE.com

我们感谢国家卫生研究院授予海东谷的赠款 (ro1ai111892) 的财政支持。我们感谢韦恩州立大学的显微镜、成像和细胞仪 (micr) 核心设施提供的技术支持。

1. 细胞播种和病毒感染
<ol><li>在病毒感染前 20-24小时, 种子 5 x 10 4 人类胚胎肺成纤维细胞或其他细胞将在生长介质中的4井11毫米交错滑块上进行检查 (dulbecco 的改性鹰介质 (dmem) 补充10% 的胎儿牛血清 (fbs))。用5% 的二氧化碳 (co2) 在37°c 下培养细胞。 注: 每口井在感染时应该有70-80% 的细胞融合。</li>
	<li>第二天, 去除生长培养基, 并在 4-10 pfu 细胞的范围内用中199中的病毒感染细胞?...

<ol>
	<li>Whitley, R., Kimberlin, D. W., Prober, C. G., Arvin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+disease.">Pathogenesis and disease.</a> Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. , (2007).</li><li>Roizman, B., Knipe, D. M., Whitley, R. J., Knipe, D. 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=Herpes+simplex+viruses.">Herpes simplex viruses.</a> Fields' Virology. , 1823-1897 (2013).</li><li>Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infected+cell+protein+0+functional+domains+and+their+coordination+in+herpes+simplex+virus+replication.">Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication.</a> World Journal of Virology. 5 (1), 1-13 (2016).</li><li>Lopez, P., Van Sant, C., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+the+nuclear-cytoplasmic+translocation+of+infected-cell+protein+0+of+herpes+simplex+virus+1.">Requirements for the nuclear-cytoplasmic translocation of infected-cell protein 0 of herpes simplex virus 1.</a> Journal of Virology. 75 (8), 3832-3840 (2001).</li><li>Kawaguchi, Y., Van Sant, C., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+1+alpha+regulatory+protein+ICP0+interacts+with+and+stabilizes+the+cell+cycle+regulator+cyclin+D3.">Herpes simplex virus 1 alpha regulatory protein ICP0 interacts with and stabilizes the cell cycle regulator cyclin D3.</a> Journal of Virology. 71 (10), 7328-7336 (1997).</li><li>Mullen, M. A., Ciufo, D. M., Hayward, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+intracellular+localization+domains+and+evidence+for+colocalization+interactions+between+the+IE110+and+IE175+nuclear+transactivator+proteins+of+herpes+simplex+virus.">Mapping of intracellular localization domains and evidence for colocalization interactions between the IE110 and IE175 nuclear transactivator proteins of herpes simplex virus.</a> Journal of Virology. 68 (5), 3250-3266 (1994).</li><li>Maul, G. G., Everett, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+location+of+PML,+a+cellular+member+of+the+C3HC4+zinc-binding+domain+protein+family,+is+rearranged+during+herpes+simplex+virus+infection+by+the+C3HC4+viral+protein+ICP0.">The nuclear location of PML, a cellular member of the C3HC4 zinc-binding domain protein family, is rearranged during herpes simplex virus infection by the C3HC4 viral protein ICP0.</a> Journal of General Virology. 75 (6), 1223-1233 (1994).</li><li>Chelbi-Alix, M. K., de The, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+virus+induced+proteasome-dependent+degradation+of+the+nuclear+bodies-associated+PML+and+Sp100+proteins.">Herpes virus induced proteasome-dependent degradation of the nuclear bodies-associated PML and Sp100 proteins.</a> Oncogene. 18 (4), 935-941 (1999).</li><li>Zheng, Y., Samrat, S. K., Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tale+of+Two+PMLs:+Elements+Regulating+a+Differential+Substrate+Recognition+by+the+ICP0+E3+Ubiquitin+Ligase+of+Herpes+Simplex+Virus+1.">A Tale of Two PMLs: Elements Regulating a Differential Substrate Recognition by the ICP0 E3 Ubiquitin Ligase of Herpes Simplex Virus 1.</a> Journal of Virology. 90 (23), 10875-10885 (2016).</li><li>Lanfranca, M. P., Mostafa, H. H., Davido, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+ICP0:+An+E3+Ubiquitin+Ligase+That+Counteracts+Host+Intrinsic+and+Innate+Immunity.">HSV-1 ICP0: An E3 Ubiquitin Ligase That Counteracts Host Intrinsic and Innate Immunity.</a> Cells. 3 (2), 438-454 (2014).</li><li>Zheng, Y., Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+three+redundant+segments+responsible+for+herpes+simplex+virus+1+ICP0+to+fuse+with+ND10+nuclear+bodies.">Identification of three redundant segments responsible for herpes simplex virus 1 ICP0 to fuse with ND10 nuclear bodies.</a> Journal of Virology. 89 (8), 4214-4226 (2015).</li><li>Samrat, S. K., Ha, B. L., Zheng, Y., Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Elements+Regulating+the+Nuclear-to-Cytoplasmic+Translocation+of+ICP0+in+Late+Herpes+Simplex+Virus+1+Infection.">Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.</a> Journal of Virology. 92 (2), e01673-e01617 (2018).</li><li>Gu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+role+does+cytoplasmic+ICP0+play+in+HSV-1+infection?.">What role does cytoplasmic ICP0 play in HSV-1 infection?.</a> Future Virology. 13 (6), (2018).</li><li>Ettinger, A., Wittmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+live+cell+imaging.">Fluorescence live cell imaging.</a> Methods Cell Biology. 123, 77-94 (2014).</li><li>Frigault, M. 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W., 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=Labeling+proteins+inside+living+cells+using+external+fluorophores+for+microscopy.">Labeling proteins inside living cells using external fluorophores for microscopy.</a> Elife. 5, e20378 (2016).</li><li>Stephens, D. J., Allan, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+microscopy+techniques+for+live+cell+imaging.">Light microscopy techniques for live cell imaging.</a> Science. 300 (5616), 82-86 (2003).</li></ol>

该协议已被用于研究1型 hsv-1 icp0 的核细胞质迁移。icp0 在1型单纯疱疹病毒感染期间遭到亚细胞贩运 (图 1)。很可能, icp0 与各种细胞通路相互作用, 在不同的位置执行不同的功能。这使得 icp0 能够在与人类主机13的拔河比赛中微调其多重功能。然而, icp0 如何以时空的方式协调多个函数还没有得到很好的研究。利用上述荧光显微镜协议, 我们开始分析 icp0 核细?...

单纯疱疹病毒 1 (hsv-1) 可引起多种轻度至重度疱疹疾病, 包括唇部疱疹、生殖器疱疹、间质角膜炎和脑炎。一旦感染, 该病毒建立在神经节神经元的终身潜伏性感染。有时, 病毒可以通过各种原因重新激活, 如发烧、压力和免疫抑制1, 导致复发性疱疹感染。感染细胞蛋白 0 (icp0) 是对裂解和潜伏性1型单纯疱疹病毒感染至关重要的关键病毒调节剂。它通过抵消宿主固有的抗病毒防御 2,3,使下游病毒基因转化.icp0 具有 e3 泛素连接酶活性, 针对几个细胞因子进行蛋白酶依赖性降解3。它还与各种细胞通路相互作用, 以调节其活动, 并随后抵消宿主抗病毒限制3。据悉, icp0 位于不同的亚细胞隔间, 因为感染进行 3,4,5。该蛋白质具有富含赖氨酸的核定位信号 (nls), 位于500至 506-6 残留物处。在早期 hsv-1 感染的早期重新合成后, icp0 立即被输入细胞核。它首先被探测到在一个称为核域 10 (nd10)7的动态核结构。icp0 的 e3 泛素连接酶活性触发 nd10 组织者蛋白、早幼粒细胞白血病 (pml) 蛋白和斑点蛋白 100 kda (sp100)8、9、10的降解。在源蛋白丢失后, nd10 核体被分散, icp0 被分散填充整个核 4,11。
有趣的是, 病毒 dna 复制开始后, icp0 从细胞核中消失。它仅存在于细胞质中, 表明在 hsv-1 感染后期发生了核-细胞质转移的4,12。dna 复制的要求意味着一种晚期病毒蛋白可能参与促进1型 hsv-1 icp04,12的细胞质移位。显然, icp0 在感染期间在不同隔间之间的贩运使 icp0 能够以时空的方式调节其与各种细胞通路的相互作用, 从而协调其多重功能, 以微调裂解和潜伏性1型 hsv-1感染13。为了更好地理解 icp0 的多功能和 icp0 功能域在整个裂解感染过程中的协调, 我们仔细地剖析了动态 icp0 移位12的分子基础。为了进行先前报道的12个机械研究, 我们应用免疫荧光染色方法, 在共聚焦显微镜下观察 icp0 亚细胞在不同感染状态下的定位。我们还开发了一个定量协议, 利用共聚焦软件分析 icp0 的核与细胞质分布。在不同的生化处理12下, 对感染 hsv-1 型 hsv 病毒的细胞在整个感染阶段进行了列表,并分析了 icp0 运动的趋势。在这里, 我们描述了记录 icp0 在1型单纯疱疹病毒感染中的易位的详细协议。我们建议将这种方法作为研究其他病毒或细胞蛋白的核与细胞质移位的一般方法, 当活体成像技术不适用时, 可以作为活体成像的替代方法:标记方法、信号强度或蛋白质丰度等问题。

<table>
	<tbody>
		<tr>
			<td>Cells and viruses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human Embryonic Lung fibroblasts (HEL Cells)</td>
			<td>Dr. Thomas E. Shenk (Princeton University)</td>
			<td></td>
			<td>HEL cells were grown in DMEM supplemented with 10% FBS</td>
		</tr>
		<tr>
			<td>HSV-1 viral Stock (Strain F)</td>
			<td>Dr. Bernard Roizman Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Invitrogen</td>
			<td>&#160;11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F0926-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium-199 (10X)</td>
			<td>Gibco</td>
			<td>11825-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4- well 11 mm staggered slide</td>
			<td>Cel-Line/Thermofisher Scientific</td>
			<td>&#160;30-149H-BLACK</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde solution(w/v) Methanol free</td>
			<td>Thermo Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher reagents</td>
			<td>BP151-1C0</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Abumin (BSA)</td>
			<td>Calbiochem</td>
			<td>CAS 9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Sigma</td>
			<td>H1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, pH7.4)</td>
			<td>Dr. Haidong Gu lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Fisher Bioreagent</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Fisher Bioreagent</td>
			<td>BP362-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>Fisher Bioreagent</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking buffer (PBS with 1% BSA and 5% Horse serum )</td>
			<td>Dr. Haidong Gu lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabiit Anti-ICP0 antibody</td>
			<td>Dr. Haidong Gu lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PML (PG-M3)-Mouse monoclonal IgG</td>
			<td>santa Cruz Biotechnology</td>
			<td>SC-966</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594-goat anti-rabbit IgG</td>
			<td>invitrogen</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-goat anti-mouse IgG</td>
			<td>invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mouting medium with DAPI</td>
			<td>Vector laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Fisher Brand</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td>Sally Hansen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica SP8</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Software</td>
			<td>Leica LAS X Application suite</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel software</td>
			<td>Microsoft Excel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HERAcell 150i CO2 incubator</td>
			<td>Thermo Scientific</td>
			<td>Order code 51026282</td>
			<td></td>
		</tr>
	</tbody>
</table>

temporal analysis of the nuclear-to-cytoplasmic translocation of a herpes simplex virus 1 protein by immunofluorescent confocal microscopy

单纯疱疹病毒 1 (hsv-1) 的感染细胞蛋白 0 (icp0) 是一种直接的早期蛋白, 含有 rg-3 泛素连接酶。它负责宿主限制性因子的蛋白酶体降解和随后的病毒基因激活。icp0 包含一个规范的核本地化序列 (nls)。它在新合成后立即进入原子核, 并主要在原子核中执行其抗主机防御功能。然而, 在感染后期, icp0 仅在细胞质中发现, 这表明在1型单纯疱疹病毒感染期间发生了核细胞质间的移位。据推测, icp0 的移位使 icp0 能够根据其在不同感染阶段的亚细胞位置调节其功能。为了描述 icp0 核细胞质迁移的生物学功能和调控机制, 我们改进了免疫荧光显微镜法, 以监测 icp0 在1型单纯疱疹病毒感染期间的贩运情况。该协议包括免疫荧光染色、共聚焦显微镜成像和核与细胞质分布分析。该协议的目标是将在时间过程中拍摄的稳态共聚焦图像调整为整个裂解感染过程中 icp0 运动的定量文档。我们提出, 该方法可以推广到定量分析核与细胞质定位的其他病毒或细胞蛋白, 而不涉及实时成像技术。

为了了解1型单纯疱疹病毒感染期间 icp0 贩运的分子基础和生物学功能, 我们采用免疫荧光显微镜法分析 icp0 在不同感染阶段的亚细胞分布。图 1显示了随着感染的进展而具有独特 icp0 定位的代表性细胞。为了量化 icp0 的核细胞质转移, 我们通过将受感染细胞分为三类来分析 icp0 相对于细胞核的分布: 核定位、细胞质定位和核加细胞质定位 (<strong class="xf...

Watch this Scientific Journal Video about Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy at JoVE.com

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

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

icp0 在1型单纯疱疹病毒感染期间经历核细胞质转移。这个事件的分子机制尚不清楚。本文介绍了共聚焦显微镜作为量化1型单纯疱疹病毒感染 icp0 运动的工具, 为定量分析 icp0 在未来的机械研究中的移位奠定了基础。

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 ...

该方法有助于回答蛋白质贩运领域有关核和细胞质转移在系统内没有光成像的关键问题。该技术的主要优点是，它可以作为研究时间蛋白运动的一般协议，不涉及光成像。20至24小时前，病毒感染种子5倍10至4个人类胚胎肺或H，纤维爆炸细胞每井到4井11毫米交错滑动和生长介质，在37摄氏度下用5%的二氧化碳进行夜间孵育。必须彻底摇动幻灯片，以确保细胞的均匀分布，同时注意避免溢出培养基。第二天，用中199替换70%至80%的汇合培养物，每个细胞包含410个斑块形成单位，并在37摄氏度的震动下放置滑梯，一小时。在孵化结束时，用新鲜生长介质替换超级细胞，将细胞返回到培养箱，以进行适当的实验感染期。对于受病毒感染细胞的免疫荧光染色，用PBS快速清洗细胞三次。接着是200微升的8至10分钟孵育，每井PBS中甲醛为4%。在固定结束时，每次洗涤用200微升PBS洗三次细胞，用每井0.2%非离子表面活性剂的100微升对细胞进行渗透，为期5至10分钟。如证明，在PBS中清洗渗透细胞三次，并在每井中加入200微升阻尼缓冲液，在室温下孵育一小时。接下来，将感兴趣的原抗体的适当浓度添加到每井中，进行两小时的室温孵育。在孵育结束时，用3，10分钟的新鲜阻尼缓冲液去除任何未绑定的原抗体，并在每一井中加入适当的二级抗体，在室温下孵育一小时，免受光线影响。在孵化结束时，如所证明的，在阻塞缓冲液中清洗细胞三次，随后在PBS中清洗一次，并在每孔中加入一滴抗淡淡安装介质，并辅以DAPI。然后在滑梯上放置盖滑，用透明指甲油密封盖斜。安装盖滑时施加温和压力可去除任何多余的安装介质，有助于确保获得高质量的图像。对于标记的受感染细胞的共生成像，选择适当的二级抗体和 DAPI 波长，将图像格式设置为 1，024 x 1，024 像素，平均行为 8。然后在 100 倍目标下的四井幻灯片上成像每个井。要计算大量单元格，请从 40 倍目标下同一井中的连续字段中获取 5 到 10 个图像。要分析ICP0的核质和细胞质分布，在共生应用软件中打开项目并选择图像。打开"数量"选项卡，然后从"工具"菜单中选择"排序感兴趣的区域"。选择"绘制线"，并在要分析的单元格上绘制一条纵向线。将显示一个直方图，显示感兴趣的蛋白质和 DAPI 的沿线的荧光强度。基于背景染色，为感兴趣的蛋白质的强度设置一个恒定阈值，用于分析每个实验中蛋白质的亚细胞分布。如果蛋白质信号平均低于核区域的阈值，但高于 DAPI 边界的阈值，则将蛋白质信号分类为主要位于细胞质内。如果蛋白质信号在整个核的阈值以上，并且超过DAPI信号的边界，则将蛋白质信号分组为细胞质，加上细胞质定位。如果蛋白质信号高于核的阈值，但平均低于DAPI信号组边界外的阈值，蛋白质信号作为核定位。最后，从不同感染时间点的每个样本中列出 200 多个受感染的细胞，并在条形图中绘制数据，以说明感兴趣的蛋白质在一段时间内的运动。如证明，该方法有助于分析感兴趣的蛋白质的核到细胞质转移。例如，受感染的细胞蛋白为零，或ICP0亚细胞分布随着病毒感染的进展而变化。为了了解感染期间ICP0贩运所需的元素，可以在不同感染阶段追踪野生型和突变型单纯疱疹病毒1感染细胞中的ICP0运动，从而可以评估ICP0在不同感染点的亚细胞分布。重要的是要记住使用单层培养，使病毒有平等的机会进入细胞，并正常化感染的多重根据病毒标题，使感染进展是可比的病毒之间和病毒内部。每次开发后，这项技术为生物学和细胞病毒学领域的研究人员通过研究细胞群体中感兴趣的蛋白质的时间亚细胞定位来探索蛋白质运动铺平了道路。

Research

JoVE Journal

Immunology and Infection

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

科研

免疫与感染

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance

表面等离子体共振检测到宿主细胞质的毒素易位

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit1.  These toxins are secreted into the extracellular milieu, but they act upon ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

神经元的初步培养体系的研究疱疹病毒潜伏期和活化

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

鼠表皮离体感染单纯疱疹病毒1型

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...