Cryosectioning skeletal muscle - I have huge problems with tissue damage (Oct/30/2008 )
I have a big problem. I have to do cryosectioning of skeletal muscle. The problem is that after my sectioning, I have big holes in my tissue slices. I have heard that this could be due to crystal formation due to freeze thaw cycles. But I have not gone through any of them that I know of.
So what do I do:
I dissect the tissue, stretch it on cork and freeze it in isopenthane on dry ice.
Then I put it in Alu-foil and keep it on dry ice and transferr it to a -80 freezer.
On the day of sectioning I precool the cryostat to -25 celsius (my cutting temp) and I put a "well/mold/plastic form" with OCT in there to cool to almost frozen. I then cut the piece of muscle I want from my sample and put it down into the OCT and transfer this to a metal bucket of isopenthane in Liquid N2.
I then let the sample freeze for some minutes and put it in the cryostat to warm up to -25 before cutting.
I have tried everything I can think of but please if any of you have any suggestions I will be grateful!
Could it be the size of the "molds" or should I warm the samples up to -25 before I put it in OCT? Would that not make it too warm and risk it thawing in the OCT?
Please help me before I go crazy...
do the sectioning very slowly to avoid tearing the tissue
Ok maybe I should clarify this a little. The holes I see are present in the middle of the the skeletal muscle cells (cross sections) and the samples do not look torn. I have heard people say that skeletal muscle samples are extremely difficult to section. But despite their advices I have not been able to resolve my problem... Thanks for your answer though it could not hurt.
I have the same problem and right now I am working on it....
I found a helpful protocol "cyosectioning" from "current ptotocols in pharmacology (1999) A.3E.1-A.3E.8", which mentions the problem of holes in tissue, and possible solution as well.
Yesterday, I followed some parts of this protocol, except the "heat sink" part. The center part of the sections are quite ok, although holes still exist at the edge of sections. I still need to improve on it.
Good luck for both of us!