Abstract
Simple SummaryThe glycocalyx (GCX) is a hydrated, gel-like layer of biological macromolecules attached to the cell membrane. The GCX acts as a barrier and regulates the entry of external substances into the cell. The function of the GCX is highly dependent on its structure and composition. Pathogenic factors can affect the protective structure of the GCX. We know very little about the three-dimensional organization of the GXC. The tiny and delicate structures of the GCX are difficult to study by microscopic techniques. In this study, we evaluated a method to preserve and label sensitive GCX components with antibodies for high-resolution microscopy analysis. High-resolution microscopy is a powerful tool because it allows visualization of ultra-small components and biological interactions. Our method can be used as a tool to better understand the role of the GCX during the development and progression of diseases, such as viral infections, tumor invasion, and the development of atherosclerosis.The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.
Highlights
The glycocalyx (GCX), a carbohydrate rich hydrogel located on the surface of cells, provides mechanical support and forms the selective barrier between the cell surface and the extracellular space [1]
This view was challenged by Ebong et al (2011), who demonstrated that the GCX of in vitro cultivated endothelial cells can be preserved by rapid slam freezing (RF) combined with freeze substitution [8]
The protocol is potentially applicable to 3D electron tomography [11]
Summary
The glycocalyx (GCX), a carbohydrate rich hydrogel located on the surface of cells, provides mechanical support and forms the selective barrier between the cell surface and the extracellular space [1]. Glycoproteins (GPs) and proteoglycans (PGs), together with covalently bound glycosaminoglycan (GAG) chains and sialic acid, are the backbone molecules of the GCX. Extending far into the extracellular space, GAG chains such as heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA) are linked and/or intertwined with specific PGs in combination with other ECM proteins [4]. Little is known about the structure and dynamics of the GCX in different cell types on an ultrastructural level. This knowledge gap is largely due to a lack of methods that allow both structural preservation and immunolabeling of GCX-specific components
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