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. Author manuscript; available in PMC: 2023 Jan 1.
Published in final edited form as: Methods Mol Biol. 2022;2413:177–182. doi: 10.1007/978-1-0716-1896-7_18

Characterization of Exosomal Surface Proteins by Immunogold Labeling

Yixin Su 1, Ashish Kumar 1, Gagan Deep 1,2
PMCID: PMC9116889  NIHMSID: NIHMS1796699  PMID: 35044665

Abstract

Exosomes are an intriguing class of nanosized vesicles (~30–150 nm in diameter) released by all cell types for intercellular communication and also for cellular metabolic waste removal to maintain cellular homeostasis. Exosomes secreted by cancer cells play an important role in supporting tumor growth and metastasis by communicating with other cells in the tumor microenvironment and distant sites. Several studies have reported that the exosomes secreted by cancer cells show distinct characteristics, including size, cargo, and surface proteins from the normal cells, and can be used as important biomarkers for diagnosis and prognosis for various cancer types. Exosomes represent many distinct biochemical and morphological characteristics than other -extracellular vesicles (EVs), including their size and surface proteins. Understanding the functional role of exosomes requires specific methods for their characterization to distinguish them from other EV and non-EV structures. Transmission electron microscopy with the immunogold labeling method allows direct detection of exosomes based on their size and specific surface protein. In this chapter, we outlined the required materials and detailed method for immunogold labeling for exosomal surface proteins and size characterization.

Keywords: Exosomes, Transmission electron microscopy, Cancer, Biomarker

1. Introduction

Exosomes, nanosized extracellular vesicles (EVs) with a size range of ~30–150 nm in diameter, are released by cells as part of their normal physiology and also during physiological and pathological stress [1, 2]. Biogenesis of exosomes includes sequential invagination of the plasma membrane that eventually leads to the formation of multivesicular bodies, which ultimately fuse with the plasma membrane to release exosomes in the extracellular environment [3]. Depending on the cell of origin, exosomes can contain many constituents of cells as cargo, including nucleic acids, lipids, metabolites, and cytosolic and cell-surface proteins. Exosomes are regarded as “snapshot” of the cells, and their cargos prominently reflect the pathophysiological state of the parental cell. Exosome role has been implicated in the removal of cellular metabolic waste and excess biomolecule to maintain cellular homeostasis [4]. Moreover, these vesicles also facilitate effective intercellular communication through the delivery of the cargos to the recipient cells. Many studies have shown that exosomes secreted by cancer cells cross talk and communicate with other cells in the tumor microenvironment and also with the distant cells to facilitate tumor growth and their metastatic spread [5].

Emerging evidence indicates that various tumor cell types secrete more exosomes, with significant changes in composition, which reflects molecular signature distinct from the exosomes secreted by corresponding normal or non-neoplastic cells [6]. Therefore, exosome cargo contents have emerged as a potential diagnostic and prognostic biomarkers to classify tumor types [7]. The wide distribution of exosomes in body fluids like blood, bile, urine, tear, and saliva allows their easy isolation and detection. However, to analyze the functional role of exosomes, it is also important to distinguish them from other EVs like microvesicles and apoptotic bodies and non-EVs (such as viruses or lipid bodies). Ongoing technological and experimental advances in the active exosome field provide valuable information regarding their morphological and biological function. The varying sizes and morphologies of exosomes can be distinguished by microscopy techniques with high resolution, such as Transmission electron microscopy (TEM). In the field of exosomes, TEM with an imaging resolution of ~1 nm has been valued for its capability to detect and characterize single exosome from other non-EV particles [8]. Furthermore, employing immunogold labeling with TEM could give information regarding biochemical properties of exosomes, including expression of specific surface marker proteins. Here, we describe a method for labeling the exosomes with primary antibodies against the specific protein on their membrane followed by gold particle-conjugated secondary antibodies, which could be observed and imaged by TEM.

2. Materials

Prepare all solutions and buffers using deionized water and analytical grade reagents. Store all reagents at 2–8 °C (unless indicated otherwise).

  1. Vortex mixer.

  2. 200-mesh copper grids (Fig. 1).

  3. Ethanol: 100%.

  4. Prepare a 4% (w/v) paraformaldehyde (PFA) solution in PBS. To make a volume of 50 mL, add 40 mL of ddH2O with stirring in a fume hood; and dissolve 2 g of PFA (Electron Microscopy Sciences) and filter the solution in a graduated cylinder using a filter paper. Add 5 mL of 10× PBS. Adjust pH to 7.4. Add ddH2O to bring the volume to 50 mL.

  5. Washing buffer (PBST): Dulbecco’s PBS without Calcium or Magnesium (DPBS) with 0.1% Tween-20.

  6. 50 mM Glycine in PBS: Dissolve 188 mg of glycine in 50 mL of PBS (1×).

  7. Blocking buffer: Bovine serum albumin (BSA) (0.5%). Dissolve 0.5 g of BSA in 100 mL of PBS (1×).

  8. 2% glutaraldehyde.

  9. Uranyl acetate (1%).

  10. Gold nanoparticle-tagged secondary antibodies (anti-mouse gold IgG or anti-rabbit gold IgG) (see Note 1).

Fig. 1.

Fig. 1

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200-mesh copper grids

3. Methods

Carry out all procedures at room temperature unless otherwise specified.

  1. Activate grids: carefully transfer grids in 100% ethanol for 20 min at room temperature (RT) (see Note 2).

  2. Fix exosomes at RT for 10 min by 4% paraformaldehyde in PBS (PFA) solution (see Note 3).

  3. Pipette fixed sample (50 μL) onto a 200-mesh copper grid with carbon-coated formvar film and incubate for 1 h at RT.

  4. Transfer the grids to PBS containing 50 mM glycine for 5 min and repeat this step 3 times (see Note 4).

  5. Transfer the grids to the blocking buffer and incubate the grids in the blocking buffer for 30 min at RT (see Note 5).

  6. Transfer the grids to primary antibody: add a primary antibody to the blocking buffer and place it on a plate overnight in a cold room (see Note 6).

  7. After the primary antibody binding, wash the grids with PBST three times for 5 min each wash. Add a gold nanoparticle-tagged secondary antibody to PBST and incubate for 2 h in the dark. Wash the grids again with PBST three times for 5 min each wash. The grids are ready to fix and stain (see Notes 7 and 8).

  8. Transfer grids to 50 μL/sample in 2.5% glutaraldehyde (GLUT) for 5 min at RT.

  9. Wash the grids seven times with PBS, 5 min each time (see Note 9).

  10. Next, the grids are placed in 50 μL of 1% uranyl acetate (w/v) for 1 min.

  11. Transfer grids to 100 μL of distilled water for 2 min.

  12. Finally, the grids are ready to be imaged using TEM (FEI Tecnai Spirit transmission electron microscope system) (Fig. 2). Representative images (with scale bar and magnification) for the exosomal surface expression of L1 cell adhesion molecule (L1CAM) and glutamate aspartate transporter (GLAST) are shown in Fig. 3 (see Note 10).

Fig. 2.

Fig. 2

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FEI Tecnai Spirit transmission electron microscope system

Fig. 3.

Fig. 3

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Presence of L1CAM (a) and GLAST (b) on the surface of exosomes was assessed by immunogold labeling. Yellow arrows indicate gold particles bound to exosomes. Magnification and scale bar for each image are shown

Acknowledgments

The authors acknowledge the Department of Defense (DOD) award W81XWH-19-1-0427 (to GD) and R01DA049267 (to GD). The authors also acknowledge Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) Cellular Imaging Shared Resource supported by NCI (P30CA012197, PI: Dr. Boris

Footnotes

1.

The size of gold particles used for immunogold labeling varies from 1 to 40 nm and can be chosen according to the type of labeling technique employed.

2.

Pick up the grid with forceps carefully and use forceps to handle grids at edges.

3.

Gloves and safety glasses should be worn and solutions should be prepared inside a fume hood.

4.

Use PBS containing 50 nmol/L glycine to saturate free aldehyde.

5.

Blocking solution, an essential step in immunogold labeling, is applied before primary antibody incubation. Blocking solution will reduce the nonspecific binding of the primary or secondary antibodies to the exosomes.

6.

The optimal amount of antibody should be decided experimentally for individual antibodies. Low-affinity antibodies require extended incubation time, and the dilution for primary antibody may be variable based on the particular antibody.

7.

Gold conjugates tend to aggregate. Generally, the temperature for incubation is kept at ambient room temperature around 16–22 °C to avoid aggregation.

8.

Include only grid/s labeled with gold nanoparticle-tagged secondary antibody (without primary antibody) as a control. Except for the absence of primary antibody, all other steps will be the same for this control group.

9.

Increasing the washing time can help to decrease the background.

10.

The higher magnification image appears to have a better background quality, i.e., fewer non-EV particles that may interfere with exosome recognition.

References

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