Visualizing endocytotic pathways at transmission electron microscopy via diaminobenzidine photo-oxidation by a fluorescent cell-membrane dye

Abstract

The endocytotic pathway involves a complex, dynamic and interacting system of intracellular compartments. PKH26 is a fluorescent dye specific for long-lasting cell membrane labelling which has been successfully used for investigating cell internalization processes, at either flow cytometry or fluorescence microscopy. In the present work, diaminobenzidine photo-oxidation was tested as a procedure to detect PKH26 dye at transmission electron microscopy. Our results demonstrated that DAB-photo-oxidation is a suitable technique to specifically visualise this fluorescent dye at the ultrastructural level: the distribution of the granular dark reaction product perfectly matches the pattern of the fluorescence staining, and the electron density of the fine precipitates makes the signal evident and precisely detectable on the different subcellular compartments involved in the plasma membrane internalization routes.

Figure 1.

Confocal optical sections of HeLa cells; the labelling with red fluorescing PKH26 was performed on cells either in suspension (a,b) or adhering to the glass cover-slip (c,d). a) Equatorial optical section of a HeLa cell showing the plasma membrane labelling. b) Superficial optical section of the same cell whose tiny membrane protrusions are also finely labelled (arrowheads in the inset). The plasma membrane fuorescence is apparent as well after labelling of adhering cells (c) in which brightly fluorescing spots are also present inside the cytoplasm. d) The higher magnification of the frame in c allows a better visualization of the intracellular fluorescing spots (arrows). Scale bars: 20 µm.

Figure 2.

Transmission electron micrographs of HeLa cells at different times after PKH26 staining; DAB photo-oxidation. a) Immediately after PKH26 treatment a fine granular photo-oxidation product diffusely borders the cell surface (thin arrows). b,c) Thirty min after PKH26 staining, many invaginations of the cell membrane (asterisk) as well as small endocytotic vesicles (arrowheads) just beneath cell surface show the photo-oxidation reaction product. d-f) After one-three h after PKH26 staining, photo-oxidation precipitates are evident in many multivesicular bodies (open arrows) as well as in multilamellar bodies inside autophagic vacuoles (arrows). These vacuolar structures often occur near the cell nucleus (N). Scale bars: 500 nm.