I'm not sure of cryosectioning, but here is a method for exosome purification from plasma that I found on SciGine. 

http://scigine.com/method.php?ID=297222

Hi Zahra I think you will struggle seeing them with a microscope. The size (at best) will be ~ 0.1 microns. Even if you do see some (and can be sure that they are not caused by background auto-fluorescence) you could never be sure that you are seeing all of the exosomes produced (maybe only seeing the largest and missing the smallest).

You could probe your sections with an antibody constitutively expressed on exosomes  (CD9 I think is one?).

You might be able to identify them based upon the fluorescence from your tagged siRNA and this ab (i.e. co-localisation of signals) but if you can see them they may appear as "punctate" spots rather than as "exosomes".

Also high powered lenses (e.g. 100x) will cause photo-bleaching faster than the low powered lenses.  Also your lens should have quite a high numerical aperture (1.5 or better) to have any chance at all.

Good luck - and let us know if you do see anything!

Gary

thanks Gary...but my purpose is not observed exosomes morphology, I just want to know exosomes enter the tissue or not? however let me know if seeing the bright spots is sufficient for this purpose? 

Hi Zahra what fluorophore is conjugated to your RNA? The problem is that several cellular components will naturally auto-fluoresce. The one which will cause problems in the blue and green channels will likely to be lipofuscin which can appear as "punctate" spots under the blue and green channels (Ex ~ 405 and 488 nm). If the fluorophore is a red one (unlikely I am guessing) you may be able to separate it from the background.

Obviously you will need a control section (tissue from a mouse that has not been transfected with the fluorescent siRNA or with vector only) to compare your sections too - so you might be able to use this to confirm that any "bright spots" you see are potentially "exosomes".

However once an exosomes fuses with an adjacent cell I imagine the exosomal content will be diffused into the host cell's cytoplasm (?). Thus any signal from the siRNA will be dispersed throughout the recipient cell so I imagine your chances of seeing a signal will in fact be zero.

The are only a few ways I can  think of doing this.

One is by using a laser dissection microscope to cut out surrounding cells and use qPCR to detect siRNA transfer. Alternatively you could try to visualise the siRNA itself in the recipient cell(s) using a RNA probe (such as Stellaris probe)

https://www.biosearchtech.com/support/education/stellaris-rna-fish

(Note: I work for LGC who distribute these probes - other RNA FISH probes may be available).

However to visualise single RNA (with probes against your fluorescence conjugate for example -  which will separate any signal from your transfected siRNA from native siRNA) requires a minimal 60x lens with a numerical aperture > 1.3 (if memory serves me right).

I'm afraid at the end of the day you will just have to try and if (and it is a big IF) you do see a signal in your transfected mouse versus your control (tumour bearing mouse not transfected with the reporter) this should be sufficient to show exosomal take up.

Sorry I can't be more help and good luck with your research,

Gary