Re: Question on Snap Freezing
|From:||Amos Brooks <email@example.com>|
Sorry for the delay ... Holiday / weekend thing.
You could use any Styrofoam container you want for the liquid nitrogen.
The main concern there is that you don't spill it on yourself.
We have a wide Styrofoam container of liquid nitrogen with a plastic
cup (500cc specimen container) of isopentane sitting inside.
When you place the cryomold into the isopentane, if you simply drop it
in there will be a slight bump where the tissue is. (I haven't figured out
why yet) If you are doing tiny biopsies, this is very problematic. What I do
is chill the bottom by holding the cryomold just on top of the isopentane.
When it freezes to the bottom (you can tell 'cause it turns white) dunk it
the rest of the way in, then after 5 - 10 seconds flip it over (upside
down). For some reason the bump doesn't form. This is very similar to simple
embedding (fixing the specimen to the bottom then finishing the freezing).
After the mold has been in the isopentane for about 5 min. we affix a
bronze chuck to it. (or whatever you use to clamp the specimen into the
cryostat) by placing the OCT on the chuck then the frozen specimen (still in
the mold) and leave it in the cryostat for a few minutes. The plastic mold
just pops off. Pull the bronze chuck one way and the plastic mold the other.
I have used the isopentane a few times. When the liquid nitrogen
evaporates, I discard the isopentane. As long as it is cold it is fine. I
wouldn't continuously dump it back into the stock and reuse it without
expecting problems though.
----- Original Message -----
From: "Stephanie Moore" <firstname.lastname@example.org>
To: "HistoNet Server" <email@example.com>
Sent: Thursday, May 24, 2001 1:32 PM
Subject: Question on Snap Freezing
> 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
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