Stephanie,
Here's what we do in our lab. I imagine that there are as many variations as
there are labs doing the procedure. I do the freezing in a plastic tri-pour
beaker. I place dry ice pellets into the bottom of the beaker and then
slowly add isopentane. The isopentane will rapidly boil off at this point so
I add it slowly and carefully to prevent splashing. Isopentane is easily
absorbed through the skin so I recommend that you wear gloves, we use
nitrile, as well as safety goggles. Also since the isopentane is very
flammable it is good to do the work under the hood both to isolate it as
well as to prevent breathing the vapors. As the isopentane cools ice
crystals will form on the outside of the container and the boiling of the
isopentane will diminish. We fill the cryomold with OCT and then orientate
the tissue in the mold so that the surface we wish to cut is facing the
bottom of the mold. Using a large pair of hemostats I can grasp the outer
edges of the mold and gently lower it onto the surface of the isopentane.
Some people advocate immediately plunging the mold below the surface of the
isopentane and I understand the merits of their argument, but I find that if
the mold floats for a few seconds on the surface the tissue freezes well and
there is a nice smooth surface at the top of the mold. As the OCT freezes
the mold will sink to the bottom of the container. I usually only freeze a
few samples at a time so I just leave the samples there until I am ready to
remove them all. Again I know some people feel that this will cause the
blocks to crack, but this has not been a problem for our lab. If we need to
store the sample I will wrap the mold in aluminum foil, place this in a
Bitran bag, and store at -80 until the tissue is needed. We reuse the
isopentane by pouring it into a container, in the hood, and allowing it to
come to room temperature before securing the cap. This prevents the pressure
buildup that will occur as the isopentane comes to room temperature. Once
the isopentane is at room temperature we secure the cap and store the
container in our flame proof cabinet. When you are ready to cut the sample
the piece of frozen OCT will simply pop out of the mold. You can place a
little additional OCT onto your chuck and then press what was the top (open
part of the mold) into the OCT. This orients the bottom of the mold as the
cutting surface. Once your tissue reaches cryostat temperature you are ready
to section. I do this so often that I'm sure I could have left out something
so feel free to contact  me if anything isn't clear. Best of luck.

John E. Tarpley 5-1-A
Associate Scientist
Amgen Inc.
One Amgen Center Drive
Thousand Oaks, CA 91320 
These Opinions are my own and not necessarily those of my employer.

-----Original Message-----
From: Stephanie Moore [mailto:smoore@brandeis.edu]
Sent: Thursday, May 24, 2001 10:33 AM
To: HistoNet Server
Subject: Question on Snap Freezing


Hello,

Recently someone responded with a nice procedure on how they snap freeze
that seems like something I could do in my lab.  I have wanted to try
snap freezing for a variety of reasons, but did not have access to
liquid nitrogen, etc.  

The method I am interested in is the isopentane/dry ice slush.  What
kind of container should this be done in?  I am also assuming this needs
to take place in a fume hood (I like to know even the "obvious"
details). I am going to order the cryomolds from Sakura.  I just fill
the mold with OCT, place the tissue inside, orient it, then lower the
mold (being held with forceps or something?) into the slush for how
long?  I didn't see that these molds were peel-away...Is their removal
from the frozen OCT difficult (do I need scissors, razor blade, etc).

How do I then attach this OCT block to the cryostat chuck (I have Leica
chucks with the concentric ring grooves) and keep the specimen in the
same orientation?  

Is the isopentane reusable?

Thanks for the info -

Stephanie Moore 
Brandeis University
Waltham, Ma
781-736-3182