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

Abstract

Source: Gachet-Castro, C. et al., Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions. J. Vis. Exp. (2021)

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....

Materials

List of materials used in this article

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