Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum

TY - JOUR

T1 - Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum

AU - Bengtsson, Dominique C

AU - Sowa, Kordai M P

AU - Arnot, David E

PY - 2008

Y1 - 2008

N2 - There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum-infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines surface antigen labeling on live cells with subsequent fixation and permeabilization, which enables antibodies to penetrate the cell and label internal antigens. The key steps of the protocol are as follows: indirect labeling of the surface antigen using a fluorescently tagged secondary antibody; fixation and permeabilization; indirect labeling of the internal antigen using a secondary antibody tagged with a spectrally distinct fluorescent dye; and detection of the differentially labeled antigens using a laser scanning confocal microscope. The protocol can be completed in approximately 7 h. Although the protocol is discussed here in the context of malaria parasite-infected cells, it can also be modified to visualize the membrane and intracellular distribution of surface and internal proteins in other eukaryotic cells.

AB - There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum-infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines surface antigen labeling on live cells with subsequent fixation and permeabilization, which enables antibodies to penetrate the cell and label internal antigens. The key steps of the protocol are as follows: indirect labeling of the surface antigen using a fluorescently tagged secondary antibody; fixation and permeabilization; indirect labeling of the internal antigen using a secondary antibody tagged with a spectrally distinct fluorescent dye; and detection of the differentially labeled antigens using a laser scanning confocal microscope. The protocol can be completed in approximately 7 h. Although the protocol is discussed here in the context of malaria parasite-infected cells, it can also be modified to visualize the membrane and intracellular distribution of surface and internal proteins in other eukaryotic cells.

U2 - 10.1038/nprot.2008.196

DO - 10.1038/nprot.2008.196

M3 - Journal article

C2 - 19180081

SN - 1754-2189

VL - 3

SP - 1990

EP - 1996

JO - Nature Protocols

JF - Nature Protocols

IS - 12

ER -