Anti mouse igg conjugated to 10 nm gold particles

Exosomes were adsorbed on the nickel grids by incubating at 37 °C for 30 min. The grids were washed in 100 µl drop of 1X PBS for 5 min and then transferred into a 100 µl drop of 0.5 M NH₄CL for 3 min. Blocking was done by placing the grid in 5% BSA for 20 min. Grids were washed and incubated overnight at 4 °C in anti-CD63 monoclonal antibody (ab8219, Abcam, UK) or anti-La monoclonal antibody (sc-166274, Santa Cruz, USA) or anti-EBNA1 monoclonal antibody (MA1-7271, Thermofisher, USA) (negative control). Each antibody was used at 1:25 dilution. Next day, the grids were washed and then incubated in secondary antibody (anti-mouse IgG conjugated to 10-nm gold particles, TAAB, UK) at 1:100 dilution for 2 hours. The grids were washed and fixed in 2.5% aqueous glutaraldehyde for 5 min, then contrasted with 2% uranyl acetate for 5 min and examined using Philips CM10 transmission electron microscope (TEM) (Philips, the Netherland) at 80 K volts.

Digoxigenin (DIG)-labeled EBER probes were diluted in hybridization buffer to a final concentration of 0.2 µg/ml⁴⁵ . For the detection of EBER1 or EBER2 individually, DIG-labeled EBER1 and EBER2 probes were used separately. Twenty microliter of the hybridization mix was added on the grids and incubated overnight at 37 °C in a humidified petri dish containing a filter paper soaked in 2X SSC. On the following day, the grids were washed in a 100 µl drop of 0.1X SSC (x6) for 5 min at room temperature and then in a 100 µl drop of 1X PBS (x6) for 5 min. The grids were incubated for 2 hours in anti-DIG antibody (clone D1-22, Sigma, Cat#D8156) at 1:50 dilution. The grids were washed in washing buffer (0.1% BSA in 1X PBS) for 10 min and then incubated with secondary antibody (anti-mouse IgG conjugated to 10-nm gold particles, TAAB, UK) for 1 hour at 1:100 dilution. Grids were washed with 1X PBS and fixed in 2.5% glutaraldehyde for 5 min. The nickel grids were jet washed with deionized water, blot dried, contrasted with 2% uranyl acetate for 5 min and blot dried again before TEM examination.