Gachet-Castro Camila, Trajano-Silva Lays Adrianne Mendonça, Baqui Munira Muhammad Abdel
Laboratory of Cell and Molecular Biology of Trypanosomatids, Department of Cellular and Molecular Biology and Pathogenic Bioagents, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
Laboratory of Cell and Molecular Biology of Trypanosomatids, Department of Cellular and Molecular Biology and Pathogenic Bioagents, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil;
J Vis Exp. 2021 Jul 10(173). doi: 10.3791/62219.
Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.
如今，可以找到各种各样的分子工具来研究寄生虫与宿主细胞的相互作用。然而，在获取能够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方面存在一些限制。此外，可用于标记锥虫的商业抗体很少。通常，针对寄生虫的多克隆抗体是在内部制备的，并且与同一物种产生的其他抗体联合使用可能更具挑战性。在此，该方案展示了如何使用在同一物种中产生的多克隆和单克隆抗体进行双重标记免疫荧光，以研究宿主细胞与病原体的相互作用。为了实现双重标记免疫荧光，首先孵育小鼠多克隆抗体，然后用与任何荧光染料偶联的二抗小鼠IgG抗体进行孵育至关重要。之后，需要额外的封闭步骤，以防止一抗的任何痕迹被下一个二抗识别。然后，在适当的时候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，使用在不同物种中产生的第三种抗体可以进行三重标记免疫荧光。此外，细胞核和肌动蛋白等结构随后可以用它们的特异性化合物或标记物进行染色。因此，这里介绍的这些方法可以针对任何一抗来源有限的细胞进行调整。
