Mouse anti tsg101

The samples were mixed with 4% paraformaldehyde in phosphate buffer (pH 7.2) in a 1:1 ratio and then applied to 200-mesh Formvar carbon-coated nickel grids. After blocking with 5% normal donkey serum and washing with PBS, the grids were incubated with either goat anti-human DPP IV (1:100, AF1180, R&D Systems, Inc., Minneapolis, MN, USA) and mouse anti-Alix (1:100, 3C4, Abnova Corp., Taipei City, Taiwan) or goat anti-DPP IV (1:100, R&D Systems, Inc., Minneapolis, MN, USA) and mouse anti-TSG101 (1:100, 4A10, Abcam, Cambridge, MA, USA) primary antibodies overnight at 4 °C. The grids were then washed with PBS and exposed to 6 nm colloidal gold-conjugated donkey anti-mouse IgG (705-195-147, Jackson Immunoresearch, West Groove, PA, USA) and 12 nm colloidal gold-conjugated donkey anti-goat IgG (715-195-150, Jackson Immunoresearch) for 1 h at room temperature (20–25 °C). After washing, the grids were re-fixed in 1% glutaraldehyde-PBS, stained with 2% uranyl acetate (pH 7), and washed with distilled water. After drying, the grids were examined using TEM (JEM-1011, JEOL, Tokyo, Japan).

For cell and exosome western blot, samples were lysed by RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA). The amount of protein was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). After protein transfer, the polyvinylidene fluoride (PVDF) membranes were blocked with 5% Difco skim milk (BD Biosciences, San Diego, CA, USA) in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h. Blots were incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: rabbit anti-CD63 (1:1,000, Abcam, cat. ab217345; Cambridge, MA, USA), rabbit anti-GM130 (1:1,000, Abcam, cat. ab52649; Cambridge, MA, USA), rabbit anti-Lamp2b (1:2,000, Abcam, cat. ab199946; Cambridge, MA, USA), mouse anti-Alix (1:1,000, Cell Signaling Technology, cat. 2171; Danvers, MA, USA), mouse anti-Tsg101 (1:1,000, Abcam, cat. ab83; Cambridge, MA, USA), rabbit anti-GAPDH (1:1,000, Cell Signaling Technology, cat. 2118; Danvers, MA, USA). Corresponding horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) (1:10,000, Pierce, Rockford, IL, USA) secondary antibodies were incubated for 1 h at room temperature. Bands were detected using an enhanced chemiluminescence (ECL) kit (Pierce, Rockford, IL, USA).

Equal volumes of EV pellets were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto PVDF membranes. The membranes were blocked with 5% milk for 1.5 h and incubated at 4°C overnight with primary antibodies: mouse anti-TSG101 (1: 1000, Abcam), rabbit anti-HSP70 (1: 1000, Abcam), mouse anti-CD63 (1: 1000, Abcam), and mouse anti-β-actin (1: 2000, Zsbio). The membranes were washed 3 times and then incubated with anti-rabbit or anti-mouse IgG antibodies (1: 4000, Zsbio) conjugated with peroxidase at room temperature for 1.5 h. The protein bands were visualized by enhanced chemiluminescence and were scanned using a luminescent image analyzer (GE Healthcare Bio-Sciences AB, Amersham Imager 600).