Dear colleagues,
We have prepared a few 6 mm sapphire discs with cells monolayers by HPF, they are in liquid nitrogen now.
These disks are too big for conventional Leica freeze substitution baskets which are used for standard 3 mm freezing hats.
In addition, we have forgotten to mark the sides of the disks and now it is practically impossible to distinguish them.
How should we organize the freeze substitution process for such objects?
Cheers,
Denis
Dear Denis,
you can do the freeze substitution in bigger vials where the discs would fit in. And later do a flat embedding with a drop of resin and mounting on dummy blocks.
To identify the side with cells you can scratch the surface of the disc a little bit with a needle or forceps before embedding (but after the osmium step).
Hope this helps a bit.
Best,
Andreas
Dear Andreas,
Thank you so much. Freeze substitution is a new area for me.
Yes, I am going to use OsO4 and it will be possible (I hope :)) to see the black cell layer on the surface. It would be more tricky for UA.
Could you describe or give a link to a good detailed protocol for embedding (Epon)? Just a flat embedding without detachment of the cells from the sapphire surface?
Cheers,
Denis
Hi Dennis,
we routinely do freeze substitution in acetone with osmium and epon embedding with the following procedure; after substitution (with the samples still in acetone with osmium) keep on ice for 1 h folowed by four washes in anhydrous acetone on ice (30 min). Next, infiltrated with Epon resin at 4°C in microtubes (on a rotary shaker at low RPM) with acetone : Epon resin mixtures (2 : 1 for 4 h; 1 : 1 overnight; 1 : 2 for 4 h; 1 : 3
for 4 h), transferred to 100% Epon resin, and left overnight on a rotary shaker at room temperature. The next day the resin is replaced with fresh 100% Epon before curing at 70°C for 3 days. at this step you can chose to move the sapphire discs to a petri-dish in a small layer of epon and place a cut-off beem capsule on top to form a stub, after one day of uring you fill up the beem capsule with 100% epon and let it harden for 3 days.
the tricky/sticky part will be replacing the resin during infiltration. Normally we use the flow-trough capsules for this (transferring the capsule with sample to a new tube with resin) but they were only developed for the 3mm system. As an alternative you could use the plastic containers developed for critical point drying (if you have a tube that they fit into) or use the 6mm flat embedding system that leica offers.
best.
Rob
Dear Andreas,
Thank you very much. It is really helpful. Is this protocol only for cells monolayer or it is suitable for tissue samples too?
Why do people use very long (from 24 to 72 h and more ) substitution time?
http://otolic.stanford.edu/documents/Recipe_book.pdf
Sorry for the idiotic questions - this area is new for me and I am trying to get as more knowledge as possible :)
Cheers,
Denis
Hi Denis,
we use it for tissue (isolated islets) and cell monolayers.
There are super short substitution protocols from Kent McDonald. They also work well for many people.
Article Freeze substitution in 3 h or less
I think most of the protocols we use have not really been compared to each other. So the long protocols might not be necessary for all samples and your question is not idiotic.
Best,
Andreas
Andreas, thank you so much. It is really very interesting. We are using two AFS in our lab now - one for OsO4 and one for UA (for CLEM). If it is really possible to dramatically decrease the substitution time, that is very good news for us.
All the best,
Denis
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