Hi all!
I need to observe the shape of epidermal cells in many Petunia mutants. I tried by fixing them with glutaraldehyde in 0.075 M cacodylate buffer (for 24-48-72h), then dehydrating in increasing concentrations of ethanol and finally I used the Critical point dryer.
The result is that cells are completely empty and "floppy", so totally unsuitable to look at the shape!
The samples fixed for 24 h are a little bit better than the others, expecially when there is some "cavity" on the sample (maybe is a bit floded) and cells maintain their normal shape.
My group has already observed them , years ago and they were perfect! The only difference was the cpd, now we changed the machine.
Any advice?
Thank you very much!
PS I don't have a CryoSEM!
Hi Martina
I don't have any experience with plants, but judging by the description, I would think that the problem may be the decompression and recompression rates. If you release the CO2 too quickly, the structure will get distorted. Try releasing the CO2 more slowly to see if it has any effect.
Alternatively, you may try using hexamethyldisilazane. After dehydrating with ethanol, you immerse the tissue in HMDS and let it evaporate. It has been used with soft animal tissue, cells, etc (see article attached)
I second that suggestion of replacement of CPD with HMDS. Works for a lot of specimens, sometimes (not always) better than CPD.
Another good method for plants - ESEM. Its ideally suited for such specimens, does not require any specimen preparation.
Yes, ESEM would definately be ok, unfortunately we do not have it in my lab.
I''ll try by HMDS, thanks!
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