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

Published: March 29, 2024

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 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). Infect LLC-MK2 cells with Trypanosoma cruzi&#1…

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

Play Video

Bu Makaleden Alıntı Yapın
A Double-Labeling Immunofluorescence Technique to Visualize Host and Pathogen Proteins in an Infected Cell. J. Vis. Exp. (Pending Publication), e22104, doi: (2024).

View Video