Hi,
I'm in the midst of troubleshooting TEM. My cells are polarized epithelia grown on Transwells. Polyester membranes are used since polycarbonate is degraded by propylene oxide, ruining the monolayer. Since they're ~7um tall, a cell thick, and both chambers are filled with fixative, I sincerely doubt diffusion constants of fixatives factor into my issue.
I've run this protocol four times now with mixed results each time. I've tried it twice with an array of conditions (acetone vs EtOH and PB vs HEPES vs Caco) and nothing has worked. I've also done it very, very poorly twice and it has worked.
I'm wondering if cacodylate buffer, which I know has arsenic, can harm cells? I usually rinse with it before fixation
Do not use cacodylate buffer before fixation, it is killing cells! You do not want to fix cells which already were dead for a while! .
Yes I totally agree with Vladimir, try rinsing with isotonic PBS (1x) at 37C for 1 min or less. Also it is unnecessary to use propylene oxide (P.O.) on cultured cells, I only use P.O. for tissue. With the cells growing on transwell membranes, I usually cut out pieces in 70% ethanol, transfer to glass vials and continue dehydration. After 3 changes in 100% ethanol (fresh pint sized unopened bottle) I just go to pure epon with catalyst (there is a little bit of ethanol left right before the change) overnight, followed by 3 x 2hr changes fresh epon with cat the next day. This will also work with the P.O.but make sure you do not expose the cells to air during changes, P.O. being much more volatile than ethanol. Good Luck.
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