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

Summary

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.

Abstract

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.

Introduction

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, such as viruses, bacteria, and protozoa, can infect most host cell types^1,2,3,4. 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 species^{6,7,8,9,10,11,12,13,14}.

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.

Protocol

1. Cell and parasite cultures

1. Grow LLC-MK2 (Rhesus Monkey Kidney Epithelial) cells from the American Type Culture Collection (CCL-7) in a 25 cm² cell culture flask containing in RPMI medium supplemented with 10% heat-inactivated FBS (Fetal Bovine Serum) and antibiotics (100 U/mL Penicillin and 100 µg/mL Streptomycin) at 37 °C in 5% CO₂ ¹⁷.
2. Infect LLC-MK2 cells with Trypanosoma cruzi (Y strain) according to a previous protocol ¹⁸.
3. Collect the supernatant of the LLC-MK2 infected cells (5 mL) in a 15 mL cell culture conical centrifuge tube and centrifuge at 500 x g for 10 minutes and 22 °C to lower cell debris. Keep the tube for 10 minutes at 37 °C to allow trypomastigotes to swim to the supernatant.
4. Collect the supernatant in a new conical tube and centrifuge at 2500 x g for 15 minutes at 22 °C. Discard the supernatant and resuspend the pellet containing the parasites in complete RPMI medium to determine cell density by counting cells in a Neubauer chamber.

2. Control immunofluorescence protocol

NOTE: Once fixed, it is possible to store plates containing coverslips at 4 °C in 1x PBS (pH 7.2) for one week. To be stored, it is important that the cells have not gone through the permeabilization step.

1. Settle LLC-MK2 cells (2 x 10⁴) in 24 well plates containing UV sterilized rounded coverslips in RPMI media for 16 hours.
2. For infected cells, add a supernatant containing T. cruzi (item 1.4) to each well in proportion (MOI 10:1) and leave for 6 h of infection. Wash coverslips containing infected and non-infected cells five times with PBS solution and fixed with 2% paraformaldehyde in 1x PBS (pH 7.2) for 10 min at room temperature (RT).
3. Wash the coverslips three times for 5 minutes each with 1x PBS (pH 7.2).
4. Permeabilize the coverslips with 0.2% IGEPAL CA-630 in 1x PBS (PH 7.2) for 10 min at RT.
5. Wash the coverslips three times for 5 minutes each with 1x PBS.
6. Incubate coverslips for 30 min at RT with the blocking solution (2% BSA in 1x PBS, pH 7.2).
7. Incubate coverslips for 30 min at RT either with mouse monoclonal anti-hnRNPA1 (dilution 1:200) or with mouse polyclonal anti-TcFAZ (dilution 1:100) antibodies diluted in blocking solution.
8. Wash the coverslips three times for 5 minutes each with 1x PBS.
9. Incubate the coverslips for 30 min at RT with goat anti-mouse IgG F (ab')2 (H+L) conjugated to Alexa Fluor 488 (1:600) together with phalloidin conjugated to Alexa 594 (1:300) to stain actin filaments (F-actin) in the host cell diluted in the blocking solution.
10. Wash three times with 1x PBS (pH 7.2) for 5 min each.
11. Apply a small amount of ProLong Gold antifade mounting reagent with DAPI medium to the surface of the slide.
12. Using forceps, gently tilt the coverslip in the mounting medium to prevent bubbles from forming. Once dry, seal the coverslip if desired.

3. Double labeling immunofluorescence protocol using monoclonal and polyclonal antibodies raised in the same host

1. Repeat steps 2.1 to 2.6 described above.
2. Incubate coverslips containing infected and non-infected cells with in-house mouse polyclonal anti-TcFAZ antibody (1:100) diluted in blocking solution for 30 min at RT.
3. Wash the coverslips three times for 5 minutes each with 1x PBS.
4. Incubate coverslips for 30 min at RT with goat anti-mouse IgG F (ab')2 (H+L) conjugated to Alexa Fluor 647 (1:600) diluted in the blocking solution.
5. Wash the coverslips three times for 5 minutes each with 1x PBS.
6. Perform the second blocking step with AffiniPure rabbit anti-mouse IgG (H+L) diluted (0.12 mg/mL) in blocking solution for 30 min at RT.
7. Wash the coverslips three times with 1x PBS (pH 7.2) for 5 min each. Then incubate with mouse monoclonal anti-hnRNP A1 IgG2b antibody (1:200) for 30 min at RT.
8. Wash the coverslips three times for 5 minutes each with 1x PBS.
9. Incubate the coverslips for 30 min with goat anti-mouse IgG2b antibody conjugated to Alexa Fluor 488 (1:600) and with phalloidin conjugated to Alexa 594 (1:300) diluted in the blocking solution.
10. Wash the coverslips three times for 5 minutes each with 1x PBS.
11. Repeat steps 2.8 to 2.10 described above.

4. Triple labeling immunofluorescence protocol with the addition of the third polyclonal antibody from different host species

NOTE: For the additional labeling, note the IgG subclasses, antibody isotypes, and follow the order of antibodies: 1. mouse polyclonal, 2. rabbit polyclonal, 3. second block, and 4. mouse monoclonal. Consider the type of lasers available in the confocal microscope to choose the correct fluorophore-conjugated secondary antibody.

1. Repeat steps 2.1 to 2.6 described above.
2. After steps 3.2 to 3.5 (washing step), start a new incubation with rabbit polyclonal antibody in blocking solution for 30 minutes at RT.
3. Wash the coverslips three times with 1x PBS (pH 7.2) for 5 minutes each.
4. Incubate the coverslips with goat anti-rabbit antibody IgG conjugated to Alexa 647 (1:600) in blocking solution for 30 minutes.
5. Wash the coverslips three times for 5 minutes each with 1x PBS.
6. Perform a blocking step with AffiniPure rabbit anti-mouse IgG (H+L) diluted (0.12 mg/mL) in blocking solution for 30 min at RT.
7. Wash the coverslips three times with 1x PBS (pH 7.2) for 5 minutes each and incubate with any mouse monoclonal IgG subclass antibody diluted in blocking solution for 30 minutes.
8. Wash the coverslips three times for 5 minutes each with 1x PBS.
9. Incubate the coverslips for 30 minutes with a specific pair of goat anti-mouse IgG subclass antibody conjugated to Alexa 594 (1:600) in blocking solution for 30 minutes.
10. Wash the coverslips three times for 5 minutes each with 1x PBS.

5. Confocal imaging acquisition

1. Analyze the immunofluorescence samples using a confocal microscope, with a 63X oil immersion objective and detect the fluorescence with a photomultiplier tube (PMT) and Hybrid detector (HyD).
   NOTE: We used the setup from the Multiuser Laboratory of Confocal Microscopy - LMMC, Ribeirao Preto Medical School, University of Sao Paulo.
2. Acquire all confocal images with separated channels. Perform image processing using Adobe Photoshop.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
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