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

This work was supported by Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo (FAPESP 2010/19547-1; 2018/03677-5) to MMAB, by Funda&#231;&#227;o de Apoio ao Ensino, Pesquisa e Assist&#234;ncia- FAEPA to MMAB and by Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior- Brasil (CAPES) - finance code 001. CG-C received a master and doctoral fellowship from CAPES and LAMT-S received doctoral fellowship from CNPq. We thank Elizabete R. Milani for confocal microscopy assistance and Dr. Dario Zamboni for providing LLC-MK2 cells (Ribeirao Preto Medical School, USP).

1. Cell and parasite cultures
<ol>
	<li>Grow LLC-MK2 (Rhesus Monkey Kidney Epithelial) cells from the American Type Culture Collection (CCL-7) in a 25 cm2&#160;cell culture flask containing in RPMI medium supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum) and antibiotics (100 U/mL Penicillin and 100 &#181;g/mL Streptomycin) at 37 &#176;C in 5% CO2 17.</li>
	<li>Infect LLC-MK2 cells with Trypanosoma cruzi (Y strain) according to a previous protocol <sup...

<ol>
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Here, we present a protocol to perform double immunolabeling in Trypanosoma cruzi infected cells using two different antibodies from the same host species. To study, with more detail, the implications of the infection, structures in the host cell such as the nucleus or cytosolic organelles can be labeled using this protocol. Also, it can be used in the post-embedding thin section immunogold labeling method. This approach helps to overcome the obstacle of having few antibodies available to study trypanosomatids a...

To study the interaction of the pathogen with the host cell at the cellular level provides essential information on the underlying causes of the disease since different groups,&#160;such as viruses, bacteria, and protozoa,&#160;can infect most host cell types1,2,3,4. It can also help develop and identify potential therapeutic targets that can slow or inhibit the growth of the pathogen. In live conditions, the produced antibodies are responsible for recognizing self-components, antigens from viruses, bacterial components or products, fungi, parasites, and others5.
For this purpose, antibodies are widely used tools, mainly for understanding the location and function of cellular structures and proteins. Several studies using multiple antibody labeling demonstrate that additional blocking steps contribute to the specificity of the immunolocalization. In addition, most described protocols use specific commercial monoclonal antibodies, including antibodies from the same host species6,7,8,9,10,11,12,13,14.
Usually, double labeling immunofluorescence uses two antibodies raised in different species to stain the cell structures of interest or the pathogens and the host cells to see the interaction between them. However, this can be a problem when no commercial monoclonal or polyclonal antibodies specific for some pathogens are available to perform the double labeling. Also, there are commercially available antibody conjugation kits, and it is possible to conjugate the primary antibodies directly to the fluorophore by a succinimidyl ester reaction15. The problem is that these kits are often expensive, and it is necessary to have enough antibodies to label them. Knowing this, we successfully developed a double immunofluorescence method using two different antibodies raised in the same species to study protein localization in Trypanosoma brucei16. However, for intracellular parasites, including Trypanosoma cruzi, this approach has not been demonstrated. Here, we show how to perform double labeling immunofluorescence to study intracellular T. cruzi parasites and the host cell using primary antibodies raised in the same species without cross-reactions. Besides this method, a triple immunofluorescence labeling has been established with the addition of the third antibody from a different species. These approaches help when the source of antibodies is limited and can be used in any cell type.

<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

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.

Here, we show how to study host-parasite interactions by immunofluorescence when the source of antibodies is limited due to the unavailability of commercial antibodies that recognize specific structures and proteins in trypanosomatids.
Among trypanosomatids, T. cruzi has one of the most complex life cycles involving various development stages between vertebrate and invertebrate hosts 19. During the T. cruzi life cycle, at an early stage of mammalian in...

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

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

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

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