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

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Gachet-Castro, Camila [1] ; Mendonca Trajano-Silva, Lays Adrianne [1] ; Baqui, Munira Muhammad Abdel [1]
Número total de Autores: 3
Afiliação do(s) autor(es):
[1] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Cellular & Mol Biol & Pathogen Bioagents, Lab Cell & Mol Biol Trypanosomatids, Ribeirao Preto, SP - Brazil
Número total de Afiliações: 1
Tipo de documento: Artigo Científico
Citações Web of Science: 0

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. (AU)

Processo FAPESP: 10/19547-1 - Caracterização funcional das proteínas gigantes de tripanossomatídeos
Beneficiário:Munira Muhammad Abdel Baqui
Modalidade de apoio: Auxílio à Pesquisa - Regular
Processo FAPESP: 18/03677-5 - Caracterização funcional das proteínas gigantes de tripanossomatídeos e a interação com a célula hospedeira
Beneficiário:Munira Muhammad Abdel Baqui
Modalidade de apoio: Auxílio à Pesquisa - Regular