A Double-Labeling Immunofluorescence Technique to Visualize Host and Pathogen Proteins in an Infected Cell

Overview

This video showcases double-labeling immunostaining with two different primary antibodies raised in the same species, binding specifically to parasite and host proteins. Furthermore, labeled secondary antibodies targeting these primary antibodies allow the distinct visualization of host and parasite proteins, facilitating the study of host-pathogen interaction.

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 Roswell Park Memorial Institute (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% carbon dioxide (CO₂).
2. Infect LLC-MK2 cells with Trypanosoma cruzi (Y strain) according to a previous protocol.
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 a 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 phosphate-buffered saline (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 ultra-violet (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 (multiplicity of infection, 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 (octylphenoxypolyethoxyethanol) 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% bovine serum albumin, BSA in 1x PBS, pH 7.2).
7. Incubate coverslips for 30 min at RT either with mouse monoclonal anti-hnRNPA1 (anti-heterogeneous ribonucleoprotein-A1) (dilution 1:200) or with mouse polyclonal anti-TcFAZ (anti-Trypanosoma cruzi flagellum attachment zone protein) (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 4',6-diamidino-2-phenylindole (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.

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