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

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.

Functions of tissue-resident eosinophils

“…With refinements in fixation methods and improvements in immunonanogold localization at the ultrastructural level 64–66 , these features can now be better studied in mouse and human eosinophils.…”

“…Improvements in techniques for electron microscopy fixation and immunonanogold localization at the ultrastructural level have enabled structural insights into the mechanisms by which granule constituents can be mobilized into the secretory apparatus 64–66 . Eosinophil granules contain an intricate membrano-vesicular network of tubules that are collapsible by treatment with the vesicular transport inhibitor brefeldin A, concurrent with the loss of cytoplasmic small vesicles and eosinophil sombrero vesicles (EoSVs) 65 , and that therefore are likely to contribute directly to the formation of cytoplasmic secretory vesicles (FIG.…”

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

“…Molecular imaging of proteins in these subcellular compartments is achieved by using a protocol that combines several strategies for ultrastructure and antigen preservation in conjunction with robust blocking of nonspecific binding sites and improved antibody penetration [13]. Visualization is accomplished with electron-dense markers (very small gold particles with 1.4 nm diameter) covalently conjugated with Fab' fragments (Fig.…”

“…The use of an improved approach for ultrastructural detection of proteins in leukocytes (pre-embedding immunonanogold EM) [13] led to the first ultrastructural identification of a vesicle-based transport of interleukin-4 (IL-4) [5] and major basic protein (MBP-1) [7] from eosinophil secretory granules. More recently, this technique located SNARES at secretory granules and vesicles [11] and identified an active intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways [14].…”

Pre-embedding Double-Label Immunoelectron Microscopy of Chemically Fixed Tissue Culture Cells

“…The protocol presented in this chapter considers only cultured cell monolayers since 3D cultures, depending on their thickness, may be more similar to tissue sections and should be processed as such for immunogold labeling for TEM. Cultures of non-adherent, floating cells can also be processed for immunogold TEM using protocols developed for tissue sections after collecting these non-adherent cells as a pellet by centrifugation [12]…”