Lead stain solution

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).

Cells in the log phase were sandwiched between copper disks and frozen in liquid propane. The frozen cells were then freeze-substituted with acetone containing 2% glutaraldehyde and 2% tannic acid. After washing with acetone, samples were further fixed with 2% osmium tetroxide in acetone and dehydrated with ethanol. Samples were then infiltrated with propylene oxide twice and with a 70:30 mixture of propylene oxide and resin (Quetol-651; Nisshin EM, Tokyo, Japan), and the propylene oxide was volatilized. Samples were transferred to fresh 100% resin, and the resin was polymerized at 60 °C. Ultrathin sections (70 nm thick) of the blocks were prepared with an ultramicrotome (Ultracut UCT, Leica Microsystems, Wetzlar Germany). The sections were placed on copper grids, stained with lead stain solution (Sigma), and observed under a transmission electron microscope (JEM-1400Plus, JOEL, Tokyo, Japan).

Small EVs isolated from RPE-1 cells were absorbed to formvar carbon coated nickel grids and immune-labelled with an anti-CD63 antibody (556019, BD Biosciences, NJ, USA), followed by 5 nM of a gold-labelled secondary antibody (British BioCell International Ltd., UK). The samples were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer. The grids were placed in 2% glutaraldehyde in 0.1 M phosphate buffer and dried, then stained with 2% uranyl acetate for 15 min and a Lead stain solution (Sigma-Aldrich). The samples were observed with a transmission electron microscope (JEM-1400Plus, JEOL Ltd., Tokyo, Japan) at 80 kV. Digital images were obtained with a CCD camera (VELETA, Olympus Soft imaging solutions GmbH, Olympus, Tokyo, Japan) [24 (link)].

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).

The fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) were used to fix the artificial vascular tissues and engrafted tissues at 4 °C. After that, the tissues were cut into 1 mm × 1 mm in size and post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer. Subsequently, the tissues were dehydrated and embedded in Epon 812 (Nisshin EM, Tokyo, Japan). Ultra-thin sections with 70 nm-thickness were prepared using an ultramicrotome (REICHERT ULTRACUT S, Leica, Wetzlar, Germany). After staining with 4% uranyl acetate and lead stain solution (Sigma Aldrich, St. Louis, MO), the tissues were observed under transmission electron microscope (JEM-1200, JEOL, Tokyo, 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.).