I am working on cells for TEM, and i follow my regular procedure , but i still gets small holes in the cell and resin around, i tryed to increase the time of infiltration but the section did not get any better.
any suggestion??
Duaa Qattan , did you start dehydration from 70%? It is not a good way. Try to use a conventional protocol with ethanol, e.g.:
30%, 50%, 70%, 90%, 95%, 100%, 100%, 100%, PO, PO, step 20 min
epon : PO
1 : 2 - 3h
1 : 1 - 3h
2 : 1 - 3h
pure - overnight
pure - to polymerization for 72h, 60C
Good luck!
This usually results from water present in the resin. If the resin is old or had a cap or two left off for awhile from individual component bottles, the components/resin will absorb water from the air.
New resin will not produce holes when polymerising.
Knowing your protocol and your specimen (attached cell culture, pellet, etc.) would help.
For now I would advise to check alcohol you use for final stage of dehydration. It should be 100% alcohol, no water. Remember, that even if label on a bottle says "100% alcohol", if a bottle was opened and used (just a few times), alcohol would absorb water from air and became 96% alcohol. Enough to create problems when embedding.
Do you use formvar coated grids? In some cases tiny holes appear in formvar film.
Denis Korneev iam using SIGMA epoxy embedding kit
fixation - Glut
dehydration 70%, 95%, 100%. 100%- propylene oxide 30 min
1:1 over night
pure over night
embedding
Duaa Qattan , did you start dehydration from 70%? It is not a good way. Try to use a conventional protocol with ethanol, e.g.:
30%, 50%, 70%, 90%, 95%, 100%, 100%, 100%, PO, PO, step 20 min
epon : PO
1 : 2 - 3h
1 : 1 - 3h
2 : 1 - 3h
pure - overnight
pure - to polymerization for 72h, 60C
Good luck!
I agree with Denis and Vladimir,
Start dehydration with 30% and use distilled 100% alcohol twice. Perform each dehydration steps for at least 10-20 min.
Use clearing step two times.
After infiltration with Pure resin 3-4 hours, use fresh pure resin for embedding.
Ravindra Thakkar 100% alcohol cannot be produced by distillation (96% max).
Vladimir Dusevich, indeed. It is pretty tricky to prepare 100% in the lab, the best way is a fresh bottle of 99.7% Absolute GR from Merck, etc.
Actually, it is possible to use molecular sieves to dehydrate 96% alcohol. Unfortunately small pieces of sieves could be embedded in a resin during TEM specimen preparation and damage diamond knife, so I stopped to use this technique long ago. I agree, to use a bottle a fresh dehydrated alcohol is the best way to prepare specimens.
We use dehydratation 50 - 70 - 90 % EtOH - anhydrid EtOH 2x change. Each incubation time is 2 min for monolayers, the anhydrid for 10min each. After that the infiltration 1:1 1h, 1:2 1h, 100% epon o/n.
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