[Histonet] Freezing small pieces of skeletal muscle tissue

From:"Dearolf, Jennifer"

Greetings, Histonetters,
I realize that freezing skeletal muscle tissue has been a topic many times before, but in going through the archives, I could not find anyone that was using the method that we are (and maybe that's the problem, since we are having issues with freezing artifact).  Thus, I am writing the list to see if anyone has any suggestions for how we might modify our methods to have better results.  
We collect our tissue fresh and mount it in 5% gum tragacanth on cork blocks (6mm thick).  To freeze the tissue, we put some isopentane in a frozen juice can (with the juice removed, of course!) and place the juice can in liquid nitrogen.  The liquid nitrogen is contained in a small, styrofoam cooler.  When the isopentane gets slushy, we freeze our samples for 60 seconds.  We then toss the frozen samples into a cryostat set at -21C.  Once the samples have warmed up and any liquid isopentane has evaporated, we wrap our samples in parafilm and store in cardboard boxes in a -70C freezer.  To cut, we move the boxes back into the cryostat and allow to wam up to      -21C for approximately an hour.  Then, we cut.  
I used this method successfully with larger pieces of muscle, but we are now attempting to freeze some mouse and guinea pig muscles (diaphragm, scalenus, rectus thoracis, and rectus abdominus), and we are getting a ton of freezing artifact.  To try to prevent the artifact, we reduced the freezing time to 20 seconds.  We also tried wrapping our samples in small pieces of liver.  Neither method seems to work consistently.  I am at my wits end.  Everytime I think we have the freezing artifact beaten, it comes back with a vengence.  
I would appreciate any advice.  I will keep track of the temperature of the isopentane, since this seems to be an important variable to control (according to the discussions in the archives).  I would like to ask, however, should the temperature be -150 or -160C when you freeze the muscle?  Thanks for your help!
Jennifer Dearolf, Ph.D.
Assistant Professor
Biology Department
Hendrix College
1600 Washington Avenue
Conway, AR 72032
(501) 450-4530 (office)
(501) 450-4547 (fax)
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