Method for preparation of small particles (or
vesicles) for electron microscopy by
ultracentrifugation
Do´nt use glass pipets or glass materials
1. Concentrate the samples to a small volume of about
200µl
2. Add 2x fixative (2x: 4% glutaraldehyde in 0.2 M
cacodylate buffer pH 7.3-7.4 mix thoroughly)
3. Transfer each about 200µl to the special centrifuge
tubes
4. Tare the centrifuge tubes in their adaptors by
adjusting the sample volume
5. Centrifuge in a “swing out rotor” at about 10°C and
with about 20% higher speed than native, unfixed
samples (sedimentation of fixed material is more
difficult)
6. Remove inserted small tubes
7. Remove supernatant carefully
8. For shipping: Overlay the pellets carefully with 0.1M
cacodylate buffer pH 7.3 – 7.4, fill up completely,
don´t resuspend and close with the corresponding
cap
Alternative preparation for shipping: Method with agarose encapsulation:








Resuspend the pellet with a very small amount (only few µls) of
buffer (0.1M cacodylate)
Melt the low gelling agarose (2-4%) by putting the tube in boiling
water or into a heating block (be sure that agarose is liquified)
Cool down the agarose to about 45°C
Heat up the samples to 37 – 40°C
Mix immediately heated samples and agarose fifty-fifty
Cool down on crushed ice for 1 hr
Remove the samples by cutting the small tubes with a razor blade
Transfer the agarose encapsulated samples to 0.1 M cacodylate
buffer (fill up cap completely)