FW: [Histonet] Re: freezing skeletal muscle

[gayle callis]

We always visually judged the correct temperature for LN2 cooled isopentane,
by using a glass beaker, although as long as you use a container to see the
following described appearance of isopentane aka 2 methylbutane when cooled,
it will work. 

We lowered a glass beaker (containing isopentane) hanging from a wire holder
(redesigned coat hanger!) into a Dewar of liquid nitrogen.  When the
isopentane becomes thick e.g. viscous, like Karo syrup or molasses, and the
bottom of container had isopentane looking a bit white, the temperature was
reached ~minus 120C. 

If we tried to test the temperature with a RT warm thermometer designed for
cold temperatures, the isopentane immediately becames less viscous, and the
temperature skewed towards warm. Doing this visually was much better than
using a thermometer.  In fact, if one cools isopentane to the point of
becoming a solid aka a glass or super-cooled liquid, you can stir the
isopentane with a long, RT metal forceps to let solvent go back to liquid
and start over.  

As for isopentane, storage in an explosion proof refrigerator or freezer is
mandatory, or store at room temperature if room is not too hot, in a
flammable storage cabinet.  This is what we have done in the past.  
It pays to be safe, and also use a fume hood to not breathe fumes from this
solvent.  

Gayle Callis 
HTL/HT/MT(ASCP)
Bozeman MT  

   

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Robert
Richmond
Sent: Thursday, November 19, 2009 11:22 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Re: freezing skeletal muscle

Galina Deyneko asks about freezing skeletal muscle.

I'm not sure that temperature control of the isopentane is too
important - it should be very slightly viscous. But do get rid of
explosive isopentane and get a non-flammable substitute - see my
previous posts on this topic.

The technique for freezing skeletal muscle is easier demonstrated than
described. Hold a small piece (maybe 5 x 10 mm) in tweezers, and coat
it with talc powder so that it appears white on the surface. Then dip
the specimen in and out of the isopentane - roughly two dips per
second - until it is frozen solid. That will eliminate the ice crystal
artifact. But you have to try it a few times before it will quite work
for you. Set aside one dead rat and one afternoon.

Bob Richmond
Samurai Pathologist
Knoxville TN

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