[Histonet] Cryostat safety question

Thu Jan 8 09:43:57 CST 2009

Very ingenious technique Claire! 

However, I would hesitate to use this with our particular cryostat as I find when doing this type of technique (keeping a button), or using any chucks which are not absolutely RT I get thick-thin sections as the tissue isn't super firmly adhered to the chucks. If I use super clean, room temp chucks my sections are 100% better quality. In fact when teaching users to use the cryostat I always make a big deal about this issue. The thick-thin issue seems to be improved somewhat since Leica revamped their chucks and now have much bigger grooves - so I am convinced firm adherance is key to quality sections.

I would be curious if anyone had any opinions on this, or experienced the same thing (thick thin sections as a result of chuck/tissue adherance). If Leica peops want to chime in about it too, I would welcome that.

Andrea

-----Original Message-----
From: Ingles Claire <CIngles <@t> uwhealth.org>

Date: Wed, 07 Jan 2009 18:38:04 
To: Histonet<histonet <@t> lists.utsouthwestern.edu>
Cc: <mari.ann.mailhiot <@t> leica-microsystems.com>
Subject: RE: [Histonet] Cryostat safety question

Andrea:
I work in a Mohs clinic where all we cut is frozen skin sections. Needless to say, we don't have 50 chucks laying around... In the morning before clinic starts we put a layer of freezing medium on chucks and put them in the cryostat to freeze. When we get specimens, we add another drop or so to the already frozen 'button' and immediately embed the tissue in it. We usually add another small drop on top after it has begun to freeze, to cover the specimen completely. Cut as normal when frozen. After done cutting all you have to do is use a forceps or other blunt object and pop the bit with the specimen in it away from the 'button' and return the chuck to the cryostat and it can be reused the rest of the day. The specimen is therefore still frozen for storage, and it has a quicker TAT. Plus you won't need nearly so many chucks, as they can be recycled almost as soon as you are done cutting. I usually keep 6-8 'buttons' in my cryostat, and our clinic can process up to 50 separate specimens a day. A word of caution. If your work area is humid sometimes a thin layer of frost can form on the surface of the 'button' and when you attempt to take sections the bit with the tissue will pop off the 'button'. All you need to do is add another drop of medium to the button and 'glue' the two back together. If you are going a while between cutting sessions, I usually store my 'buttons' upside(mountant side) down on one of the cryostat surfaces. It doesn't seem to develop the frost layer. Useful if you have tiny specimens.
Hope my verbose explanation is helpful. Feel free to e-mail if you have any questions or are confused about my explanation. 

Claire Ingles
Madison WI

________________________________

From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Andrea Hooper
Sent: Wed 1/7/2009 5:40 PM
To: Histonet
Cc: mari.ann.mailhiot <@t> leica-microsystems.com
Subject: [Histonet] Cryostat safety question

The discussion on microtome safety begs me to ask a cryostat question ....

We have a Leica CM3050 cryostat and love it!

How are people (and perhaps only those in research do this) removing
their tissue from the chucks for future use? We often just section a
few slides worth then put the block at -80 deg C for future studies.
Needless to say, it's the most dangerous part of our day.

So what are your suggestions for removing tissue from a chuck (and
melting it isn't really a viable option)?

Thanks in advance,
Andrea
--

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