I am planning a HPF hybrid (pre-light chemical fixation) technique on attached cells for HM-20 post embed IEM. My plan is to lightly fix the cells for 1 hr (4% PF 0.5% Ga 0.1M HEPES with 5mM MgCl2), rinse in 0.1M HEPES 3.5% sucrose with 5mM MgCl2 (243 mOsmols), then scrape and pellet the cells. I will re-suspend the pellet in 2% agarose (in the cells media minus serum) just before HPF. My question is, has anyone used HEPES as an IEM buffer, and at what concentration (molarity)? I have read that a slightly hypotonic solution is better for IEM and I believe the cultured cells are isotonic at 300 mOsmols. Also for IEM, is there really a difference between 4% PF and 2% PF?
Dear Michael,
I found this your request just a minute ago.
Needless to state that your question can not easily be answered.
Maybe you have gotten meanwhile either valuable answers or other information about how HEPES will work as a buffer vehicle for primary fixation of tissue prior to HPF.
For now I would like to point you only to a former thread
() which you can find @ https://www.researchgate.net/post/Alternatives_to_formalin_fixation
and which contains lot of literature references and perhaps some answer concerning HEPES too (cf. "HOPE" technique).
I certainly will be informed of an answer in this thread, so perhaps we can discuss this your request more in detail (I am bit short in time now). Good luck, best wishes and regards,
Wolfgang
Wolfgang,
Yes we have made progress with HPF IEM HM20 using HEPES on lightly fixed attached cells. My next plan is to go with PHEM buffer, and HPF the pre-fixed cells in 1% agarose in PHEM with 3% sucrose. We are also investigating different solvents other than acetone (ETOH) for the AFS part, with and without osmium. We are striving for good labeling with better morphology and are making progress. Will keep you posted.
sincerely,
Michael Delannoy
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