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 ...

In this protocol, two antibodies from the same host can be used together in the immunofluorescence assay to study host cell and pathogen interactions. Since there are only few commercial antibodies available to recognize specific cell structures and proteins in parasites, this protocol can be very useful. This is an easy-to-perform double-labeling immunofluorescence protocol that uses polyclonal and monoclonal antibodies raised in the same species.Many researchers may be unaware that this is possible. This approach helps when the source of antibodies is limited. It can be used to detect the pathogens in the host cell proteins in infected host cells and can also be applied to free living organisms.This is a simple protocol, require one blocking step between the first and second pair of antibodies. The procedure will be demonstrated by Dr.Camila Gachet-Castro and PhD student Lays Trajano-Silva from my laboratory. Three days after infecting LLC-MK2 cells with Trypanosoma cruzi, collect the supernatant from the infected cells in a 15 milliliter cell culture conical tube.Centrifuge the sample to lower cell debris, then place the tube at room temperature to allow the trypomastigotes to swim to the supernatant. After 10 minutes, collect the supernatant in a new conical tube, then centrifuge the sample and discard the supernatant before resuspending the pellet containing the parasites in complete RPMI medium. Add LLC-MK2 cells in 24-well plates containing UV sterilized rounded coverslips and allow the&m to settle for 16 hours.Then to infect the cells, add supernatant containing T.Cruzi to each well and incubate for six hours. Next, wash the coverslips containing the infected and non-infected cells five times with PBS, then fix the cells with 2%paraformaldehyde in PBS. After 10 minutes, wash the coverslips three times for five minutes each with PBS and then permeabilize the coverslips with non-ionic detergent for 10 minutes.After three five-minute PBS washes, incubate the coverslips in blocking solution for 30 minutes, followed by another 30-minute incubation with either a mouse monoclonal or polyclonal antibody. After PBS washes, incubate the coverslips for 30 minutes with secondary antibody in blocking solution and phalloidin to stain actin filaments in the host cell. Then wash the coverslips with PBS three times again before applying a small amount of anti-fade mounting reagent with DAPI medium to the surface of the slide.Using forceps, gently tilt the coverslip in the medium to prevent bubble formation. For double labeling, after incubating the coverslips in blocking solution as demonstrated previously, incubate them in the mouse polyclonal antibody for 30 minutes. Next, wash the coverslips three times for five minutes each with PBS before incubating them with the secondary antibody for 30 minutes.Then following three PBS washes, perform a second blocking step with AffiniPure rabbit anti-mouse IgG diluted in blocking solution. After 30 minutes, wash the coverslips with PBS again before incubating them with the mouse monoclonal antibody for another 30 minutes. Next, after three PBS washes, incubate the coverslips with goat anti-mouse IgG 2B antibody and phalloidin.Then following three PBS washes, apply anti-fade mounting reagent as demonstrated previously. For triple labeling, after blocking the coverslips and incubating with the mouse polyclonal antibody, followed by goat anti-mouse antibody, start a new incubation with rabbit polyclonal antibody in blocking solution for 30 minutes. Then after three PBS washes, incubate the coverslips with goat anti-rabbit antibody for 30 minutes.Next, following three PBS washes, perform a blocking step with AffiniPure rabbit anti-mouse antibody diluted in blocking solution for 30 minutes. Then wash the coverslips again before incubating with any mouse monoclonal IgG subclass antibody for 30 minutes. Following three PBS washes, incubate the coverslips for 30 minutes with a specific pair of goat anti-mouse IgG subclass antibodies.After incubation, wash the coverslips three times for five minutes each with PBS. Analyze the immunofluorescent samples using a confocal microscope with a 63X oil immersion objective and detect the fluorescence with a photomultiplier tube and hybrid detector. Then acquire all confocal images with separated channels and perform image processing using Adobe Photoshop.These confocal microscopy images show results with the control experiments of infected and non-infected cells highlighting the specificity of the antibodies in the host cell and the internalized parasite. The mouse polyclonal antibody anti-Trypanosoma cruzi FAZ recognized the T.cruzi giant protein in the flagellum attachment zone at the parasite flagellum, but not in the host cell. Distribution of the heterogeneous nuclear ribonucleoprotein A1 in the nuclei was observed using a commercial mouse monoclonal antibody that recognizes only the host mammalian cells, but not the parasite.Also, the host and parasite nuclei and the parasite kinetoplasts were stained with DAPI and host F-actin was stained with phalloidin conjugated to Alexa 594, confirming the specificity of the antibodies. Double-labeling immunofluorescence shows the protein distributions in the host and the parasite analyzed by confocal microscopy. The labeling suggested no cross-reaction between antibodies using this methodology.In summary, the protocol described here to study host-pathogen interactions presents a basic and elaborate technique that can be adapted to any combination of antibodies. This approach makes immunofluorescence cost effective and can help study the localization of two or more proteins of interest when few antibodies are available.

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

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

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

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 ...