Looking for all kinds of information on handling, safety, starting material, solution preparation, grid choice...
Any information is appreciated. Thank you.
Hello
- For the grids, 400 mesh formvar coated grids. I don't bother about metal (classically copper or nickel) but nickel is magnetic so avoid magnetic tweezers.
- You manipulate the grid with a very thin tip tweezers (non-magnetic).
- 2% PTA solution pH 7, protected from the light and keep at 4°C. I suggest also to filter or sonicate to remove aggregates and also de-gas.
- a gridbox.
For safety, PTA has nothing to do so far with Uranyl Acetate (radioactive).
One way to do staining is:
- put a drop (minimum 5 micro-litters) of your liposome solution on parafilm on the bench, then put the grid on it, formvar-side in contact with the liquid, for several minutes. As it is a small volume, cover it to avoid drying.
- prepare a drop of your PTA solution (the drop can be bigger than 5 micro-litters).
- take the grid with the tweezers and remove the excess of liquid with filter paper by the side of the grid and put it immediately on the PTA drop for few seconds (5-10 seconds).
tip: move the grid slowly at the surface of the paper as this one absorbs the liquid to keep its absorption power.
Don't let dry at this step!
- after few seconds staining, remove the excess of liquid with filter paper by the side of the grid (same tip) and let dry.
- storage in the gridbox.
It is ready for MET observation!
remark: if your liposomes are in high salty buffer you may wash with water before PTA staining.
You should get images like the one joined. PTA pass through the membrane so you can see the multilayers.
good luck with you work!
Hello Nicolas Barois ,
Thank you very much for this detailed procedure, I will try this, and such beautiful image you have!
Just a couple of details: Does it make a difference if I purchase crystalline PTA to make the 2% aq solution or if I purchase the 10% solution and dilute that? (silly question, I know, but I really can't find any comparison for this). These are the only options I can find for purchasing. The MSDS for the crystalline PTA requires high protective gear, so 10% solution would be easier to handle on a daily work basisi IF it gives the desired results.
Again thank you very much.
Yes silly question ;)
I haven't done the PTA solution myself. I found it in the freezer when I took the job. So it looks like it is very stable over long time, just filter or sonicate and de-gas time to time when needing it.
To answer the question, I will make a comparison with the osmium tetroxide, which is very toxic, we are using a lot in EM. I stopped to buy its crystal form, avoiding the dilution step, and buy now ready-made solution.
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