Jenn,
I'm sure you'll get some good advice after the weekend from people who do 
this all the time. My initial thought is, that your specimen should go from 
isopentane directly to -70C, not -20C, unless you cut it then. When you 
transfer the tissue to -70C, ice ctystal artifact will form, when tissue 
freezes slowly from -20 to -70.
Just a thought...

Katri
Katri Tuomala
Hamilton, Ontario, Canada

----- Original Message ----- 
From: "Dearolf, Jennifer" 
To: 
Sent: Friday, June 09, 2006 3:20 PM
Subject: [Histonet] Freezing small pieces of skeletal muscle tissue


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!

Sincerely,
Jenn

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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