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Nat Protoc. 2014 Oct;9(10):2382-94. doi: 10.1038/nprot.2014.163. Epub 2014 Sep 11.

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

Author information

1
1] Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil. [2] Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
2
Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
3
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

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.

PMID:
25211515
PMCID:
PMC4204927
DOI:
10.1038/nprot.2014.163
[Indexed for MEDLINE]
Free PMC Article

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