Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages

Summary

Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0° C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37° C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.3∼3 μm thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 200∼1,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0° C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37° C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stercomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.