Name: double labeling immunofluorescence using antibodies from the same species to study host-pathogen interactions
Uploaded: 2021-07-10T14:00:00
Description: Watch this Scientific Journal Video about Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions at JoVE.com

这项工作得到了圣保罗保护儿童基金会（FAPESP 2010/19547-1; 2018/03677-5）向MMAB，由Apoio ao Ensino基金会，Pesquisa e Assistência-FAEPA到MMAB以及由巴西高级国家福利协会（CAPES）支持 - 财务代码001。CG-C获得了CAPES的硕士和博士奖学金，LAMT-S获得了CNPq的博士奖学金。我们感谢Elizabete R. Milani的共聚焦显微镜协助和Dario Zamboni博士提供LLC-MK2细胞（Ribeirao Preto医学院，USP）。

1. 细胞和寄生虫培养
<ol><li>在37°C下在37°C下在5%CO2 17中加入RPMI培养基的25 cm2细胞培养瓶中培养来自美国型培养物（CCL-7）的LLC-MK2（恒河猴肾上皮）细胞，该培养瓶中含有10%热灭活FBS（胎牛血清）和抗生素（100 U / mL青霉素和100μg/ mL链霉素）。</li>
	<li>根据先前的方案用 克氏锥虫 （Y菌株）感染LLC-MK2细胞 18。</li>
	<li>将LLC-MK2感染细胞（5mL...

<ol>
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在这里，我们提出了一种方案，使用来自同一宿主物种的两种不同抗体在 克氏锥虫 感染的细胞中进行双重免疫标记。为了更详细地研究感染的影响，可以使用该协议标记宿主细胞中的结构，例如细胞核或细胞质细胞器。此外，它可用于后包埋薄片免疫金标记方法。这种方法有助于克服可用于研究锥虫和其他寄生虫的抗体很少的障碍。
此外，我们的方案显示细胞内寄生...

为了在细胞水平上研究病原体与宿主细胞的相互作用，提供了有关疾病根本原因的基本信息，因为不同的群体，如病毒，细菌和原生动物，可以感染大多数宿主细胞类型1，2，3，4。它还可以帮助开发和识别可以减缓或抑制病原体生长的潜在治疗靶点。在活体条件下，产生的抗体负责识别自身成分、病毒中的抗原、细菌成分或产品、真菌、寄生虫等5。
为此，抗体是广泛使用的工具，主要用于了解细胞结构和蛋白质的位置和功能。几项使用多抗体标记的研究表明，额外的阻断步骤有助于免疫定位的特异性。此外，大多数描述的方案使用特定的商业单克隆抗体，包括来自同一宿主物种的抗体6，7，8，9，10，11，12，13，14。
通常，双标记免疫荧光使用在不同物种中培养的两种抗体来染色感兴趣的细胞结构或病原体与宿主细胞以查看它们之间的相互作用。然而，当没有针对某些病原体的特异性商业单克隆或多克隆抗体可用于执行双重标记时，这可能是一个问题。此外，还有市售的抗体偶联试剂盒，并且可以通过琥珀酰亚胺酯反应将一抗直接偶联到荧光团15。问题在于，这些试剂盒通常很昂贵，并且有必要有足够的抗体来标记它们。知道了这一点，我们成功地开发了一种双免疫荧光方法，使用在同一物种中培养的两种不同抗体来研究布鲁氏锥虫中的蛋白质定位16。然而，对于细胞内寄生虫，包括克氏锥虫，这种方法尚未得到证实。在这里，我们展示了如何进行双重标记免疫荧光，以使用在同一物种中培养的一抗来研究细胞内克氏锥虫寄生虫和宿主细胞，而不会发生交叉反应。除了这种方法之外，还建立了三重免疫荧光标记，并添加了来自不同物种的第三种抗体。当抗体来源有限且可用于任何细胞类型时，这些方法会有所帮助。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 - IgG2b antibody</td>
			<td>Life technologies, USA</td>
			<td>A21141</td>
			<td>Goat anti-mouse</td>
		</tr>
		<tr>
			<td>AffiniPure Rabbit anti-mouse IgG (H+L)</td>
			<td>Jackson Immunoresearch, USA</td>
			<td>315-005-003</td>
			<td>Anti-mouse antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 - IgG F (ab&#39;)2 (H+L) antibody</td>
			<td>Life technologies, USA</td>
			<td>A11017</td>
			<td>Goat anti mouse</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 IgG1 antibody</td>
			<td>Life technologies, USA</td>
			<td>A21125</td>
			<td>Goat anti-mouse</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 - IgG F (ab&#39;)2 (H+L) antibody</td>
			<td>Life technologies, USA</td>
			<td>A21237</td>
			<td>Goat anti-mouse</td>
		</tr>
		<tr>
			<td>Anti-hnRNPA1 antibody IgG2b</td>
			<td>Sigma-Aldrich, USA</td>
			<td>R4528</td>
			<td>Mouse antibody</td>
		</tr>
		<tr>
			<td>anti-TcFAZ (T. cruzi FAZ protein) antibody</td>
			<td>Our lab</td>
			<td>In-house</td>
			<td>Mouse antibody</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich, USA</td>
			<td>A2153-10G</td>
			<td>Albumin protein</td>
		</tr>
		<tr>
			<td>Detergent Igepal CA-630</td>
			<td>Sigma-Aldrich, USA</td>
			<td>I3021</td>
			<td>Nonionic Detergent</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco, Thermo fisher scientific, USA</td>
			<td>12657-029</td>
			<td>Serum</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco, Thermo fisher scientific, USA</td>
			<td>15140-122</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Phalloidin Alexa Fluor 594</td>
			<td>Life technologies, USA</td>
			<td>A12381</td>
			<td>Actin marker</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade with DAPI</td>
			<td>Life technologies, USA</td>
			<td>P36935</td>
			<td>Mounting media reagent</td>
		</tr>
		<tr>
			<td>RPMI 1640 1X with L-glutamine</td>
			<td>Corning, USA</td>
			<td>10-040-CV</td>
			<td>Cell culture media</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T4049-100ML</td>
			<td>Bioreagent</td>
		</tr>
	</tbody>
</table>

double labeling immunofluorescence using antibodies from the same species to study host-pathogen interactions

如今，有可能找到广泛的分子工具可用于研究寄生虫 - 宿主细胞相互作用。然而，获得识别寄生虫中特定细胞结构和蛋白质的商业单克隆或多克隆抗体存在一些局限性。此外，很少有商业抗体可用于标记锥虫。通常，针对寄生虫的多克隆抗体是内部制备的，与同一物种中产生的其他抗体结合使用可能更具挑战性。在这里，该协议展示了如何使用在同一物种中培养的多克隆和单克隆抗体来执行双标记免疫荧光以研究宿主细胞和病原体的相互作用。为了实现双标记免疫荧光，首先孵育小鼠多克隆抗体至关重要，然后与与任何荧光染料偶联的二级小鼠IgG抗体一起孵育。之后，需要一个额外的阻断步骤，以防止任何痕量的一抗被下一个二抗识别。然后，在适当的时间将小鼠单克隆抗体及其与不同荧光染料偶联的特异性IgG亚类二抗添加到样品中。此外，可以使用在不同物种中培养的第三种抗体进行三重标记免疫荧光。此外，核和肌动蛋白等结构随后可以用其特定的化合物或标记物染色。因此，这里提出的这些方法可以针对一抗来源有限的任何细胞进行调整。

在这里，我们展示了当抗体的来源由于无法识别锥虫体中特定结构和蛋白质的商业抗体而受到限制时，如何通过免疫荧光研究宿主 - 寄生虫相互作用。
在锥虫中，克氏锥虫具有最复杂的生命周期之一，涉及脊椎动物和无脊椎动物宿主之间的不同发育阶段19。在克氏锥虫生命周期中，在哺乳动物感染的早期阶段，变环锥体突变体通过涉及寄生虫和...

Watch this Scientific Journal Video about Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions at JoVE.com

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

在这里，该协议描述了如何使用在同一物种中培养的一抗来研究宿主 - 病原体相互作用来执行双重标记免疫荧光。此外，它可以包括来自该协议中不同宿主的第三种抗体。这种方法可以在任何细胞类型和病原体中进行。

使用来自同一物种的抗体进行双标记免疫荧光研究宿主 - 病原体相互作用

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

在该协议中，来自同一宿主的两种抗体可以在免疫荧光测定中一起使用，以研究宿主细胞和病原体的相互作用。由于只有少数商业抗体可用于识别寄生虫中的特定细胞结构和蛋白质，因此该协议可能非常有用。这是一种易于执行的双标记免疫荧光方案，使用在同一物种中培养的多克隆和单克隆抗体。许多研究人员可能没有意识到这是可能的。当抗体来源有限时，这种方法会有所帮助。它可用于检测受感染宿主细胞中宿主细胞蛋白中的病原体，也可用于游离生物体。这是一个简单的方案，需要在第一对和第二对抗体之间进行一个阻断步骤。该程序将由我实验室的Camila Gachet-Castro博士和博士生Lays Trajano-Silva演示。用克氏锥虫感染LLC-MK2细胞三天后，将感染细胞的上清液收集在15毫升细胞培养锥形管中。离心样品以降低细胞碎片，然后将试管置于室温下，以使锥孢子虫游到上清液中。10分钟后，将上清液收集在新的锥形管中，然后离心样品并丢弃上清液，然后将含有寄生虫的沉淀重悬在完整的RPMI培养基中。将LLC-MK2细胞加入含有紫外线灭菌圆形盖玻片的24孔板中，并允许&m沉降16小时。然后感染细胞，向每个孔中加入含有T.Cruzi的上清液并孵育六小时。接下来，用PBS洗涤含有受感染和未感染细胞的盖玻片五次，然后用PBS中的2%多聚甲醛固定细胞。10分钟后，用PBS清洗盖玻片三次，每次五分钟，然后用非离子洗涤剂使盖玻片透化10分钟。在三次五分钟的PBS洗涤后，将盖玻片在封闭溶液中孵育30分钟，然后用小鼠单克隆或多克隆抗体再孵育30分钟。PBS洗涤后，用封闭溶液中的二抗和鬼臼素孵育盖玻片30分钟，以染色宿主细胞中的肌动蛋白丝。然后用PBS再次洗涤盖玻片三次，然后将少量带有DAPI培养基的防褪色安装试剂施加到载玻片表面。使用镊子轻轻倾斜介质中的盖玻片，以防止形成气泡。对于双重标记，如前所述，在封闭溶液中孵育盖玻片后，将它们在小鼠多克隆抗体中孵育30分钟。接下来，用PBS洗涤盖玻片三次，每次五分钟，然后用二抗孵育30分钟。然后在三次PBS洗涤后，用在封闭溶液中稀释的AffiniPure兔抗小鼠IgG进行第二次阻断步骤。30分钟后，再次用PBS洗涤盖玻片，然后用小鼠单克隆抗体再孵育30分钟。接下来，在三次 PBS 洗涤后，用山羊抗小鼠 IgG 2B 抗体和苷孵育盖玻片。然后在三次PBS洗涤后，应用防褪色安装试剂，如前所述。对于三重标记，在封闭盖玻片并与小鼠多克隆抗体孵育后，接着是山羊抗小鼠抗体，在封闭溶液中开始与兔多克隆抗体进行新的孵育30分钟。然后在三次PBS洗涤后，用山羊抗兔抗体孵育盖玻片30分钟。接下来，在三次PBS洗涤后，用在封闭溶液中稀释的AffiniPure兔抗小鼠抗体进行阻断步骤30分钟。然后再次洗涤盖玻片，然后与任何小鼠单克隆IgG亚类抗体孵育30分钟。在三次PBS洗涤后，用一对特定的山羊抗小鼠IgG亚类抗体孵育盖玻片30分钟。孵育后，用PBS清洗盖玻片三次，每次五分钟。使用带有63X油浸物镜的共聚焦显微镜分析免疫荧光样品，并使用光电倍增管和混合检测器检测荧光。然后，使用单独的通道采集所有共聚焦图像，并使用 Adobe Photoshop 执行图像处理。这些共聚焦显微镜图像显示了受感染和未感染细胞的对照实验的结果，突出了宿主细胞中抗体和内化寄生虫的特异性。小鼠多克隆抗体抗克氏锥虫FAZ在寄生虫鞭毛的鞭毛附着区识别出T.cruzi巨蛋白，但在宿主细胞中没有。使用商业小鼠单克隆抗体观察细胞核中异质性核糖核蛋白A1的分布，该抗体仅识别宿主哺乳动物细胞，而不识别寄生虫。此外，用 DAPI 染色宿主和寄生虫细胞核以及寄生虫动力学体，并用与 Alexa 594 偶联的蛋白染色宿主 F - 肌动蛋白，证实了抗体的特异性。双标记免疫荧光显示受试者体内的蛋白质分布和通过共聚焦显微镜分析的寄生虫。标记表明使用这种方法的抗体之间没有交叉反应。总之，这里描述的用于研究宿主 - 病原体相互作用的方案提出了一种基本且复杂的技术，可以适应任何抗体组合。这种方法使免疫荧光具有成本效益，并且当抗体很少可用时，可以帮助研究两种或多种感兴趣蛋白质的定位。

Research

JoVE Journal

Immunology and Infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

动态活细胞成像的宿主 - 病原体相互作用的研究使用的光学陷阱

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

科研

免疫与感染

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

使用的EpiAirway模型，用于描述长期宿主 - 病原体相互作用

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

成像InlC的分泌由细菌病原体调查感染蜂窝<em&gt;单核细胞增生李斯特氏菌</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

Isolation of Double Negative ÃƒÅ½Ã‚Â±ÃƒÅ½Ã‚Â² T Cells from the Kidney

从肾脏双重否定αβ性T细胞的分离

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...