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

Nature Protocols volume 3, pages 1990–1996 (2008)Cite this article

Abstract

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 ∼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.

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Figure 1: Illustrated flowchart of the protocol for the simultaneous visualization of surface membrane and intracellular antigens of malaria parasite-infected human red blood cells.
Figure 2: Confocal laser scanning fluorescence micrographs of a preparation of dual-labeled P. falciparum–infected erythrocytes.
Figure 3: Confocal images of fluorescent-labeled human red blood cells (uninfected), reacting with a mouse monoclonal antibody against an unknown ∼75-kDa red blood cell antigen, detected with an Alexa 488 (green)–labeled human anti-mouse IgG antibody.

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Acknowledgements

This work was supported by a Niels Bohr Foundation Visiting Professorship to D.E.A. Thanks to Dr Anja Jensen for providing the VAR4 antibody and Dr Jana McBride for providing the mouse monoclonal antibody 2.1 against the 75-kDa human erythrocyte antigen.

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Authors and Affiliations

  1. Department of International Health, University of Copenhagen and Department of Infectious Diseases, Immunology and Microbiology, Centre for Medical Parasitology, Copenhagen University Hospital (Rigshospitalet), CSS Øster Farimagsgade 5, Building 22, PO Box 2099,

    Dominique C Bengtsson, Kordai M P Sowa & David E Arnot

  2. 1014, Copenhagen K, Denmark

    Dominique C Bengtsson, Kordai M P Sowa & David E Arnot

  3. Institute for Immunology and Infection Research, School of Biological Sciences, Ashworth Laboratory, Edinburgh University, West Mains Road, Edinburgh, EH93JT, Scotland, UK

    Dominique C Bengtsson, Kordai M P Sowa & David E Arnot

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  1. Dominique C Bengtsson
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  2. Kordai M P Sowa
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  3. David E Arnot
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Contributions

D.C.B. developed and validated the protocol and wrote the manuscript. K.M.P.S. and D.E.A. developed the protocol and wrote the manuscript.

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Correspondence to David E Arnot.

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Bengtsson, D., Sowa, K. & Arnot, D. Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum. Nat Protoc 3, 1990–1996 (2008). https://doi.org/10.1038/nprot.2008.196

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