Em 14830ruby2

MDA‐MB‐231‐luc‐D3H2LN cells were cultured at 35°C, 37°C, or 39°C for 1 day and fixed with 2% paraformaldehyde and 2% glutaraldehyde (GA) in 0.1 M phosphate buffer (pH 7.4) at room temperature (RT). The samples were incubated at 4°C for 30 min, and fixed with 2% GA in 0.1 M phosphate buffer at 4°C overnight. They were washed three times with 0.1 M phosphate buffer and post‐fixed with 2% osmium tetroxide in 0.1 M phosphate buffer at 4°C for 1 h. The samples were dehydrated in graded ethanol solutions (50%, 70%, 90%, and 100%) as follows: 50% and 70% for 5 min each at 4°C, 90% for 5 min at RT, and three changes of 100% for 5 min each at RT. The samples were embedded in Quetol‐812 (Nisshin EM, Tokyo, Japan) and polymerized at 60°C for 48 h. The polymerized resins were sectioned at 70 nm using an ultramicrotome (Ultracut‐UCT; Leica, Wetzlar, Germany). The sections were mounted on copper grids, stained with 2% uranyl acetate at RT for 15 min, washed with distilled water, and stained with Lead stain solution (Sigma‐Aldrich, St. Louis, MO, USA) at RT for 3 min. The grids were observed using transmission electron microscopy (JEM‐1400Plus; JEOL) at an acceleration voltage of 100 kV. Digital images were acquired with a CCD camera (EM‐14830RUBY2; JEOL).

Ciona ovaries were fixed in 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) (Distilled EM grade, Electron Microscopy Sciences, Hatfield, PA, USA) in 0.1M phosphate buffer (PB) pH 7.4 at 4 °C for 1h. After washing, the samples were dehydrated and infiltrated with a 50:50 mixture of ethanol and resin, transferred to a fresh 100% resin (LR white, London Resin, Berkshire, UK), and polymerized by an ultraviolet polymerizer. Ultra-thin sections of the polymerized resins were mounted on nickel grids and incubated with anti-PEP51 antibody, followed by 15nm gold particle-labelled secondary antibody. The grids were placed in 2% GA in 0.1 M PB and dried, then stained with 2% uranyl acetate for 15 min and a Lead stain solution (Sigma-Aldrich, Tokyo, Japan). The samples were observed under a transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan) at 100 kV. Digital images were obtained with a CCD camera (EM-14830RUBY2, JEOL). Immunoelectron microscopy was carried out by Tokai Electron Microscopy, Inc. (Nagoya, Japan).

To determine whether binding AuNPs to AG1478 affected AuNP uptake, lysosomal uptake of AuNP was measured by TEM. HSC-3 cells were cultured with 60 nm AuNPs (50 nM), both alone and as a mixture of 60 nm AuNP (50 nM) and AG1478 (0.5 μM). The samples were fixed with 2% paraformaldehyde and 2% glutaral aldehyde (GA) in 0.1 M phosphate buffer (PB) pH 7.4 at incubation temperature and put into a refrigerator for 30 min to lower the temperature to 4°C. Thereafter, they were fixed with 2% GA in 0.1 M PB at 4°C overnight. After fixation, the samples were washed three times with 0.1 M PB for 30 min each and postfixed with 2% osmium tetroxide (OsO₄) in 0.1 M PB at 4°C for 1 h. The samples were dehydrated in an alcohol gradient, then transferred to a resin (Quetol-812; Nisshin EM Co., Tokyo, Japan), and polymerized at 60°C for 48 h. The polymerized resins were sectioned at 80 nm with a diamond knife, using an ultramicrotome (Ultracut UCT; Leica, Vienna, Austria), mounted on copper grids, stained with 2% uranyl acetate for 15 min, washed with distilled water, and secondary stained with lead stain solution (Sigma-Aldrich Co., Tokyo, Japan) for 3 min. The grids were visualized by a TEM (JEM-1400Plus; JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were taken with a CCD camera (EM-14830RUBY2; JEOL Ltd.).

Spheroid cells transfected with negative or CLDN2 siRNA were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (PB) at 4 °C overnight. The preparation and electron microscopic analysis of samples were performed by Tokai Electron Microscopy (Nagoya, Japan). The fixed samples were postfixed with 2% osmium tetroxide in 0.1 M PB at 4 °C for 2 h, followed by dehydration, infiltration, and polymerization. Then, the polymerized resins were ultra-thin sectioned at 70 nm with a diamond knife using an ultramicrotome (Ultracut UCT, Leica, Vienna, Austria) and the sections were mounted on copper grids. They were stained with 2% uranyl acetate at room temperature for 15 min and then washed with distilled water followed by being secondary-stained with Lead stain solution (Sigma-Aldrich) at room temperature for 3 min. The grids were observed by a transmission electron microscope (JEM-1400Plus, JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images were taken with a CCD camera (EM-14830RUBY2, JEOL).

For TEM, the EV-containing samples were absorbed into carbon-coated copper grids and stained with a 2% phosphotungstic acid solution (pH 7.0) for 10 s. The grids were observed by TEM (JEM-1400 Plus; JEO L Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were obtained using a CCD camera (EM-14830RUBY2; JEOL Ltd., Tokyo, Japan).