[Histonet] Freezing small pieces of skeletal muscle tissue

[Lewis, Sarah]

Jen, 


	This is the protocol we send to outside institutions.  I hope it
will help. You mentioned you wait until the 2-methylbutane/isopentane
gets slushy... This we do not do.  I listen for the "boiling" sound to
stop(so to speak). Your solution should be clear.. Not cloudy. You will
start to see little white crystals form... This is when you put your
tissue in the 2-methylbutane suspended in liquid nitrogen. A 0.5x0.5x1.0
pc of muscle should be in for 30sec. Tissue any smaller, cut your time
down by 5 sec according to size. Hold tissue in cryostat or -20/-40
until tissue is dry. Wrap with foil and store in -80.  If you have any
other questions just give me a call. I hope this helps with your
problem.

	Take Care, 
			Sarah

Sarah E. Lewis 
Histotechnician
Mendell Neuromuscular Lab
Childrens Research
Center for Gene Therapy 
700 Childrens Dr Rm WA3112
Columbus OH 43205
(614)-722-2204
LewisS <@t> ccri.net
    

-----Original Message-----
From: Katri Tuomala [mailto:katri <@t> cogeco.ca] 
Sent: Sunday, June 11, 2006 7:29 PM
To: Dearolf, Jennifer; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Freezing small pieces of skeletal muscle tissue

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...





----- Original Message -----
From: "Dearolf, Jennifer" <Dearolf <@t> hendrix.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
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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