Hi all,
So I will isolate my microvesicles from my cells soon, and will have to fix them for electron microscopy.
my only concern here is that I got a protocol of fixation for biopsy pieces.
Thus I could modify some of its steps so it fits for the microvesicles I guess...
If someone here has a protocol that works, I would be happy to try it out.
meanwhile I will try and ask someone who has experience and drop an answer if I get one.
Thanks,
Good day to all -
Treat them as a cell suspension. You can find a lot of protocols on the web, both for TEM and SEM (which one will you use?).
http://web.path.ox.ac.uk/~bioimaging/Documents/fixation_standard.pdf
https://www.google.com/webhp?sourceid=chrome-instant&rlz=1C1OPRB_enUS553US553&ion=1&espv=2&ie=UTF-8#q=cell+suspension+fixation+electron+microscopy&*
Sample preparation procedures will vary depending on the type of electron microscopy you are planning to do and the questions you are asking. If you are planning to look at your microvesicles using negative stain, they may not need fixation before hand at all. Otherwise, I suggest fixing them by adding an equal volume of 8% Paraformaldehyde and 0.2% Glutaraldehyde in PHEM buffer. Are you planning to embed them in resin and work at room temperature or plunge freeze them for cryo EM?
Thank you for your answers,
sorry I forgot to mention the type of electron microscopy I will be using , it will be the TEM.
to be honest I'm still quiet new in all this , so what I am planning to do is getting pictures of my microvesicles by electron microscopy nothing more.
I got suggested to fix them by glutaraldehyde 2,5% then add cacodylate buffer 0,1M and that's it.
I've also seen in some articles that they first fix their microvesicles with paraformaldehyde.
Dear @Pylore Ulaval: any good news? How you've prepared our samples (especially which fixative/fixatives) and what was the outcome in TEM so far? Did you - last but not least - use either only" 2.5% glutaraldehdye + 0.1M cacodylate buffer and that's it" (which might decrease buffer molarity to a certain amount), did you use also other buffers than cacodylate (e.g. PO4 or HEPES or PIPES) or only "paraformaldehyde" (plain, or buffered FA ), have you found any differences, morphological or technical difficulties in saving your microvesicles to the TEM-grid ?
Thank you for your efforts and sharing with us your acknowledged information.
@Wolfgang I used 2.5% glutaraldehdye + 0.1M cacodylate buffer. the TEM worked great! I could see the microvesicles but they were dark yet well defined vesicles compared on what was on articles over the internet ( I'll post some pictures if I can).
I did not use other buffers than cacodylate. I might test other samples soon actually so I might try some different techniques.
Hi Pylore,
Would you mind posted what fixation worked best for your exosomes?
thanks,
MD
Michael Delannoy ,
I work with microvesicles (MVs), here's my protocol :
1- add 1mL of glutaraldehyde 2,5 % in a microtube
2- add MVs suspension (100 µL) in the solution
3- leave the sample for 4-16 hour in a cold place
4- the sample was then centrifuged at 21 000g/20* min. at 4⁰C. ( *this centrifugation is for MVs isolation, for exosomes do your usual protocol.)
5- discard the supernatant and add 1mL of Cacodylate 0,1M buffer
6- Centrifuge at 21 000g/20* min. at 4⁰C.
7- Repeat step 5-6 three times.
8- Resuspend 100µL of Cacodylate 0,1M Buffer on the MVs pellet.
9- Store the sample in a cold environment until the day for TEM
10- (This step is not done by me) Samples were stained with 3% uranyl acetate
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