Hello,

It is better to use TEM from fresh sample but you can also use stored sample under appropriate conditions. For short-term, exosomes can be stored at 4°C for up to 1 week. For long-term storage, exosomes can be stored at -20°C or -80°C. When storing exosomes long-term, it is important to consider if they will need to be thawed more than once.

Thawing the preparation on ice should reduce the level of morphological changes.

This report may provide some insight,

Article Effect of pH, temperature and freezing-thawing on quantity c...

I would not freeze exosomes at all for this kind of analysis. It would be better to fix them and load on TEM grids immediately and then store in the fridge before TEM observation. However beware that this storage time should also be as short as possible.

Hi,

In terms of negative stain TEM I often see this "erythrocyte" like morphology (also seen in the literature). This is most likely due to a collapse of the structure due to lack of support by the contrast solution. So it might be worth playing around with different contrast solutions in order to get the best possible structural support but at the same time obtain the best possible resolution of the vesicle. Uranyl Acetate is commonly used, phospho tungestic acid is a useful solution for many types of samples - commonly used for viruses (and therefor also for exosomes). but produces less contrast than UA. Ammonium molybdate is also used frequently - which has also been used to negatively stain thawed thin cryosections of fixed cells. Sometimes I also combine this with methyl cellulose in order to protect the sample from the electron beam but this has an effect on the resolution.

kind regards

ketil

They are applicable but be aware that u my get exosomes with bigger size in diameter due to freeze/defreeze, also -20 is not a good choice to store exosome for a month.

Good luck