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Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes

Nature Protocols volume 9, pages 2382–2394 (2014)Cite this article

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Abstract

Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.

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Figure 1: Immunogold electron microscopy technique.
Figure 2
Figure 3: Pre-embedding immunonanogold electron microscopy procedures.
Figure 4: Example of excessive growth of gold particles (arrowheads) and background formation observed in the cytoplasm of a human eosinophil leukocyte after labeling for perilipin 2 (PLIN2/ADRP) and incubation with silver enhancement components for 20 min, which was too long.
Figure 5: Examples of pre-embedding immunonanogold labeling of human leukocytes.
Figure 6: Human leukocyte subcellular sites immunolabeled for different antigens using pre-embedding immunoEM.
Figure 7: Example of immunogold labeling of a mouse tissue (skin).

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Acknowledgements

We gratefully acknowledge the skillful assistance of T. Sciutto and K. Pyne (Electron Microscopy Unit, Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School). We acknowledge K. Bonjour for careful assistance with the illustrations (Figs. 1 and 2) used in this paper. This work was supported by US National Institutes of Health (NIH) grants R37AI020241, R01AI022571 (P.F.W., A.M.D., E.M. and R.C.N.M.), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) grants 305983/2011-3, 477475/2013-2 (R.C.N.M.) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil; R.C.N.M.).

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Authors and Affiliations

  1. Department of Biology, Laboratory of Cellular Biology, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil.,

    Rossana C N Melo

  2. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Rossana C N Melo & Peter F Weller

  3. Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Ellen Morgan, Rita Monahan-Earley & Ann M Dvorak

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  1. Rossana C N Melo
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Contributions

R.C.N.M., A.M.D. and P.F.W. designed the experiments. R.C.N.M. carried out sample preparation, immunolabeling and electron microscopic processing and analyses. E.M. and R.M.-E. provided technical assistance. E.M. carried out ultramicrotomy. All authors wrote the paper.

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Correspondence to Rossana C N Melo.

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Melo, R., Morgan, E., Monahan-Earley, R. et al. Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes. Nat Protoc 9, 2382–2394 (2014). https://doi.org/10.1038/nprot.2014.163

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