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.
Highlights
TEM is an outstanding tool for providing a comprehensive view of the interior of a cell at the nanometer scale, and it continues to have a crucial role in biological research and diagnostic pathology[1,2,3,4,5,6]
Postembedding immunogold electron microscopy (EM) has been used since the 1970s, and it is mostly convenient for detecting abundant antigens[9]
We demonstrated that CD9 is abundant on the surface of eosinophil leukocytes, presenting the first EM data of the ultrastructural immunolocalization of CD9 in human eosinophils, which represents a novel insight into the organization of the antigen presentation complex of these cells[24]
Summary
TEM is an outstanding tool for providing a comprehensive view of the interior of a cell at the nanometer scale, and it continues to have a crucial role in biological research and. Our protocol uses very small gold particles (1.4 nm in diameter) covalently conjugated with Fab fragments (Fig. 1), which are only one-third the size of a whole IgG molecule These very small probes improve antibody penetration and provide more quantitative labeling of antigenic sites[8,18], with access to membrane microdomains. In addition to optimal epitope preservation, this protocol provides excellent access of the antibodies to cell subcompartments This is important for the identification of small molecules such as those of the immune signaling system that are not usually detected by postembedding EM. Because vesicular traffic from secretory granules underlies secretion in different cell types, including leukocytes, a challenge to understanding this secretory pathway in more detail has been the identification of granule-derived products within transport vesicles This protocol enabled the first ultrastructural identification of a vesiclebased transport of interleukin-4 Beyond its use in these fields, the technique is applicable to the immunolocalizaton of a wide range of proteins within varied cell types
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