Anti cd63 ab59479

Optimal concentrations of purified EVs derived from expanded CD133⁺ cells were loaded onto 300-mesh niquel/formvar-coated grids (Electron Microscopy Science), and after 1 h of absorption to the formvar, the grids were fixed with 4% paraformaldehyde (Electron Microscopy Science) for 10 min. For immunogold staining, the grids were previously immersed in blocking buffer (PBS/2% BSA) for a block/permeabilization step for 30 min before labeling with the primary antibodies at the appropriate dilution for 1 h at room temperature (1 : 200 anti-CD63, ab59479, Abcam; 1 : 100 anti-CD105, 555.690, BD Bioscience; and 1 : 100 anti-CD31, 555.444, BD Bioscience). After rinsing, the grids were incubated with a specific antibody conjugated to 15 nm gold nanoparticles (1 : 20 25.233, Electron Microscopy Science) for 40 min at room temperature. The grids were postfixed in 2.5% glutaraldehyde (Sigma-Aldrich) for 10 min and then stained for contrast using 2% uranyl acetate for 10 min. The samples were examined on a transmission JEOL JEM-1011 electron microscope (JEOL Ltd.) at the Electron Microscopy Facility (Universidade Federal de Santa Catarina (UFSC) and Universidade Federal do Paraná (UFPR)) operating at an acceleration voltage of 80 kV.

EV‐rich fractions (8–9) were subjected to transmission electron microscopy to assess the presence and morphology of EV. A drop (3 µL) of sample was adhered to pre‐coated formvar/carbon/glow discharged 100 mesh copper‐palladium grids for 20 min. Grids were fixed with 2% formaldehyde (Science Services) in 0.1 m phosphate buffer for 20 min, washed with sterile water and stained with 2% uranyl acetate for 5 min. For immunogold labeling, after fixation, the grids were immersed in 50 × 10⁻³ m glycine in PBS for 15 min and blocked for 10 min with 10% fetal calf serum (FCS) in PBS. Without rinsing, the grids were immediately placed into the primary antibody (1:200 diluted in 5% FCS in PBS, anti‐CD63 ab59479, Abcam) for 30 min at RT. As control, some of the grids were not exposed to the primary antibody. Grids were rinsed with 0.5% FCS in PBS and incubated with 12 nm colloidal gold‐AffiniPure goat‐anti‐mouse IgG secondary antibody (1:20 diluted in 5% FCS in PBS, 115‐205‐146, Jackson Immunoresearch) for 30 min at RT. The grids were rinsed with PBS and post‐fixed in 1% glutaraldehyde (Science Services) for 5 min. After rinsing in distilled water, the grids were stained using 2% uranyl acetate for 5 min. Grids were examined using electron microscopy (FEI Tecnai G2 Spirit BioTWIN operating at 120 keV equipped with an Olympus‐SIS Veleta CCD Camera).