Hello,
I am currently trying to do cryo-sections of Xenopus laevis oocytes in order to visualize the cell membrane, but I'm having trouble to get good sections.
That's what I tried so far (from what I found in the literature):
- thickness between 3-30um
- fixing the oocytes in PFA and washing with methanol or leaving the oocytes unfixed
- cutting temperature between -23C - -18C
- carefully drying the cells on paper vs embedding the cells directly after taking them out the storage solution
- embedding singe oocytes vs whole ovaries
I am currently embedding the oocytes in OCT (is it a problem that it is a bit older, expired in 2020?)
None of the methods is working at the moment and I did not notice a huge difference between the different conditions.
My main problem is that the oocytes seem to fall out of the OCT sheet after the sectioning, in addition the inner part of the oocyte seems to crumble (which isn't too problematic for me since I'm interested in the membrane)
Does anyone has suggestions what I'm doing wrong?
Thanks a lot!
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