What is the difference between fresh frozen tissues and tissues frozen in tissue freezing medium?
Hello everyone,
I have some mouse tumors that I am going to harvest. The vet who performs the surgery will place them in an isotonic saline solution, which they will be in for a brief time (< 1 hour) before I can get them back to the lab. I have read many protocols but I still don't know which is best. Right now I see my main assays being H&E, so what I care most about is preserving the morphology and tissue structure. How long could they be left in the saline solution in the fridge at 4 °C? I assume though that they would be better frozen as soon as possible. I will embed them in tissue freezing medium for cryosectioning. Is it better to store tissues by themselves in -80 °C or first embed them in tissue freezing medium. How important is the freezing rate depending on whether the tissue is embedded in tissue freezing medium or not? The cryomicrotome system that I use has a small area on it that gets down to around -60 °C. If I place the specimen in a plastic mold or a metal mold and freeze it in that small area, is that fast enough? What about just placing it in the -80 °C freezer to freeze? Finally, are snap-freezing protocols or sucrose cryoprotection protocols something I should look into in conjunction with tissue freezing medium embedding? Thank you.
Update (11/12/2015). The undergraduate student who performed the cryosectioning made the mistake of freezing some of the initial samples straight after removal from the saline solution at -80 C. We examined both mouse tumor and mouse spleen. For the pre-frozen samples, we decided to do fixation followed by cryoprotection in 15% and 30% sucrose on those samples anyways, finally embedding in OCT compound. The spleen had many tears, especially at 10 micron thickness, specifically near the center. A 20 micron thickness gave us the best results, with much less tearing and even some whole slices. The tumors appeared to hold up better despite the initial freezing compared to the spleens. 10 microns did give us more smaller tears running through the tumors, and 20 microns again gave us the best.
With the fresh samples, they cut great, but it seemed that they started to crack a short time after they were adhered to the slide. Is there anything we can do to avoid this?
