Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx

Abstract

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.

Keywords: confocal laser scanning microscopy; freeze-substitution; glycocalyx; high-pressure freezing; immuno-electron microscopy; pre-embedding immunogold labeling.

Figure 1

Morphology and antigenicity of the cell surface in THP-1 cells after formalin fixation. (A,B) CSLM images showing immunofluorescence single labeling for GCX markers: (A) CD44 (green pseudocolor) and (B) CS (green pseudocolor) with (C) the corresponding negative control with omitted primary antibody. Note the CD44-labeling and scattered clusters of CS close to the plasma membrane (arrows). (D) WGA staining (red pseudocolor). (A–D) DAPI fluorescence stains for nuclear DNA (blue pseudocolor) Scale bar: (A–D) 5 µm.

Figure 2

Flowchart showing the workflow of the combined HPF/FS/rehydration and pre-embedding immunogold labeling method for TEM. Insert on the left side shows well-preserved intra- and pericellular ultrastructure of THP-1 cells after processing for immuno-TEM. Note the dimension of the GCX. The dashed lines mark the border of the nucleus to cytoplasm and cytoplasm to GCX. Insert on the right shows a higher magnification of the GCX structures with immunogold labeling for CD44 (arrowheads). N, nucleus; C, cytoplasm; GCX, glycocalyx. Scale bar: 1000 nm.

Figure 3

Immuno-TEM images of the ultrastructure of the GCX of THP-1 cells. The electron micrographs show embedded THP-1 cells at lower magnification for a morphological overview of the cellular and extracellular structures (A,C,E) and at higher magnification for a detailed intra- and pericellular morphology of individual THP-1 cells (B,D,F) after standard osmication (A,B), optimized aldehyde/UAc substitution (C,D), following rehydration (E,F). Note the better preservation of cellular membranes after FS with osmium (A,B) and consistently well-preserved ultrastructure of the GCX after osmication, optimized FS, following rehydration. N, nucleus; C, cytoplasm; GCX, glycocalyx. Scale bar: (A,C,E) 2500 nm; (B,D,F) 1000 nm.

Figure 4

Pre-embedding immunogold labeling for GCX antigens of THP-1 cells after HPF, osmium-free FS, and rehydration. (A–F) TEM micrographs showing immunogold labeling for CD44 (A–C) and CS (D–F) in the GCX of THP-1 cells. (A,D) GCX structures labeled by antibodies directed against either CD44 (A) or CS (D), indicated by 30 nm or 25 nm gold particles, respectively. (B,E) Same images as in (A) and in (D), with gold particles graphically highlighted in magenta to illustrate the distribution pattern and labeling density of CD44 and CS in the GCX. (C,F) Magnification of the highlighted region in B and E showing the localization of colloidal gold particles in the original size. N, nucleus; C, cytoplasm. Scale bar: (A–F) 1000 nm.

Figure 5

Flowchart showing the workflow of combined HPF/FS/rehydration, and immunofluorescence labeling for CLSM. Insert on the left side shows THP-1 cells with WGA staining that exclusively binds to GCX structures. Note that the WGA staining is not restricted to the plasma membrane but extends to the pericellular region. Insert on the right presents a merged image of immunofluorescence labeling for CS (green pseudocolor), DAPI staining (blue pseudocolor), and WGA staining (red pseudocolor). The dashed lines label the nuclear-to-cytoplasmic and cytoplasmic-to-GCX borders to visualize the extension of GCX into the pericellular region of the THP-1 cell. Scale bar: 5 µm.

Figure 6

CLSM images of immunofluorescence labeling of CD44 and CS after HPF, FS, and rehydration. (A,B) CLSM images showing immunofluorescence single labeling for GCX markers: CD44 (A; green pseudocolor) and CS (B; green pseudocolor). Note that the CD44-labeling (arrows) and scattered clusters of CS (arrows) extend beyond the plasma membrane into the GCX. (C,D) Additional co-staining for DAPI (blue pseudocolor) and WGA (red pseudocolor). The last images on the right side show colocalization of CD44 or CS immunofluorescence and WGA staining extending into the pericellular region. Scale bar: (A–D) 5 µm.

Figure 7

Control experiments. (A) Immuno-EM of negative controls with omitted primary antibodies. Note the very rare presence of single gold particles (arrow). (B) THP-1 growth medium processed for TEM exhibits no GCX-like structures. (C,D) CLSM images of negative controls with omitted antibodies for CD44 (C) and CS (D), co-staining with DAPI, and colocalization of unspecific signals for conjugated secondary antibodies. Note the weak cytoplasmic immunofluorescence background for the secondary antibody for CS (D). Scale bars: (A,B) 1000 nm; (C,D) 10 µm.