Summary

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

Published: July 10, 2021
doi:

Summary

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。

Abstract

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。

Introduction

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1234。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67891011121314

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。

Protocol

1. 细胞和寄生虫培养 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)。 根据先前的方案用 克氏锥虫 (Y菌株)感染LLC-MK2细胞 18。 将LLC-MK2感染细胞(5mL…

Representative Results

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…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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)。

Materials

Alexa Fluor 488 – IgG2b antibody Life technologies, USA A21141 Goat anti-mouse
AffiniPure Rabbit anti-mouse IgG (H+L) Jackson Immunoresearch, USA 315-005-003 Anti-mouse antibody
Alexa Fluor 488 – IgG F (ab')2 (H+L) antibody Life technologies, USA A11017 Goat anti mouse
Alexa Fluor 594 IgG1 antibody Life technologies, USA A21125 Goat anti-mouse
Alexa Fluor 647 – IgG F (ab')2 (H+L) antibody Life technologies, USA A21237 Goat anti-mouse
Anti-hnRNPA1 antibody IgG2b Sigma-Aldrich, USA R4528 Mouse antibody
anti-TcFAZ (T. cruzi FAZ protein) antibody Our lab In-house Mouse antibody
Bovine Serum Albumin (BSA) Sigma-Aldrich, USA A2153-10G Albumin protein
Detergent Igepal CA-630 Sigma-Aldrich, USA I3021 Nonionic Detergent
Fetal Bovine Serum (FBS) Gibco, Thermo fisher scientific, USA 12657-029 Serum
Penicillin Streptomycin Gibco, Thermo fisher scientific, USA 15140-122 Antibiotic
Phalloidin Alexa Fluor 594 Life technologies, USA A12381 Actin marker
ProLong Gold antifade with DAPI Life technologies, USA P36935 Mounting media reagent
RPMI 1640 1X with L-glutamine Corning, USA 10-040-CV Cell culture media
Trypsin-EDTA solution Sigma-Aldrich, USA T4049-100ML Bioreagent

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Cite This Article
Gachet-Castro, C., Trajano-Silva, L. A. M., Baqui, M. M. A. Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions. J. Vis. Exp. (173), e62219, doi:10.3791/62219 (2021).

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