1. Pre-check EVs concentration: EVs samples should be check by negative stain & NTA for checking morphology & concentration. (Over 5X1010 EVs/mL)

2. Plasma coating on Ni-carbon grid

3. Cover Ni grid on 5 μL sample droplet, 10-15 min

4. 1% BSA/PBS wash, 1min X3

5. 1% BSA/PBS as blocking buffer for 30 min

6. 1% BSA/PBS + o.o1-0.02% Digitonin as permeabilization buffer for 2 min

7. 1% BSA/PBS wash, 1min X3

8. 1st Ab, 10~20-fold dilution in 1% BSA/PBS, 90 min at RT or 4℃ O/N

9. 1% blocking buffer wash, 1min X3

10. 2nd Ab, 10-fold dilution in 1% BSA/PBS, 60 min at RT

11. 1% BSA/PBS wash, 1min X4

12. PBS wash, 1min X3

13. DDW wash 1min X2

14. 2% UA for negative stain, 1 min

15. Air dry