A longer suggestion--RE: [Histonet] frozens from lung-tissue

[Cheryl R. Kerry]

That helps a bit--

Nitrogen and most other reagents dehydrate as well as freeze if they come in
direct contact with the wet tissue.  Think 'freeze-dried coffee'. Isopentane
is less harsh--which is why it's often the contact chemical being chilled by
nitrogen in a double-boiler manner. Dehydration on a small scale is inherent
with the method.  If you cover your tissue with OCT before freezing, it will
protect it from this minor dessication while freezing.  

With a tissue as 'wet' as lung, you also work against a freezing artifact if
you let it freeze too slowly, it will crack if you freeze it too quickly.
The forces of the structure of the water expanding when freezing will create
significant fissures--both big enough to be seen with the naked eye and
microscopic ones--so your pathologist does have a legitimate beef. As with
most things in life, too little or too much isn't the best way.

Putting the freezing weights on the tissue sometimes mush it and distort
tissue relationships.  The less mechanical pressure you can put on the
tissue, the better.  This leads to the next suggestion.

There are disposable molds--plastic ones--that come just for freezing OCT
embedded tissues.  They are in several different sizes. You can buy (or have
made) a heat-sink bar that is bored out in the correct size for the
embedding mold to fit in. It doesn't have to be a purchased heat sink bar--a
small plate of aluminum or any heat-conductive metal will help pull the temp
down quickly (think cupcake pan).   You put a little alcohol in the bored
out bar or on the metal to keep the mold from sticking (like PAM kitchen
spray for frozens!) then add the mold (cupcake paper liner) then add a
little OCT, your tissue and more OCT with the chuck inverted on top into the
wet OCT (like a cream-filled cupcake in the making--the lung is the center).

Any time you let OCT freeze then add more, you've created a weak junction
that is more likely to fracture when cutting.  Whenever possible add more
OCT to the prior application before it's completely solid to make a stronger
union and prevent chunking off the tissue when you cut.  

Once it's frozen you pull the plastic mold out of the bar and the mold from
the OCT frozen tissue (cupcake paper with the now-done cupcake in it out of
the tin and strip off the paper ready, mmmmMMmm, making myself hungry), cut
the sloppy bits away, mount on the cryostat head and cut.  

The heat sink bar would come from the manufacturer of the cryostat--or
perhaps your physical plant guys could make one if you showed them a picture
(our guys were always game to try a new project).  Try Michael Vadeboncoeur
from Fisher --he responded earlier about the glove liners and hand cream for
frozen hands) for the molds: michael.vadeboncoeur <@t> thermofisher.com

Sorry this was so long--you can get samples of the molds and try it for
nothing lost but a little time and effort.  I hope this helps!!

Cheryl  

-----Original Message-----
From: Gudrun Lang [mailto:gu.lang <@t> gmx.at] 
Sent: Wednesday, November 21, 2007 12:36 PM
To: 'Cheryl R. Kerry'
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: AW: [Histonet] frozens from lung-tissue

The reason is more the antipathy paired with unfamiliarity with this
reagens. I tried it with a small amount of liquid nitrogen in a plasticbox,
where I placed the (metal) mold into it, with the tissue and the OCT until
it cracks most times. I thought it would be the fastest way of freezing, but
the result didn't suit. So I am not very ambitious to do it again.

The doctor always speaks about "over-freezing" of the edges and a overall
bad morphology.

Thank you for the hint with the cryobath.

Gudrun Lang
 
-----Ursprüngliche Nachricht-----
Von: Cheryl R. Kerry [mailto:tkngflght <@t> yahoo.com] 
Gesendet: Mittwoch, 21. November 2007 19:12
An: gu.lang <@t> gmx.at; histonet <@t> lists.utsouthwestern.edu
Betreff: RE: [Histonet] frozens from lung-tissue

Gudrun--I suspect this isn't quite what you were looking for but have you
guys tried a cryobath?  It's a little blue countertop 'freezer' unit that
has well of pre-chilled isopentane in it, always cold enough to snap freeze
without the drama of liquid nitrogen.  It is about the size of a big toaster
and is ready to go 24/7 with minimal maintenance (temp chart and periodic
cleaning).  It wasn't overly expensive from what I remember for the great
quality of the end results.  We mounted the tissue on a pre-frozen
OCT-covered chuck, covered it with liquid OCT, lowered it into the cryobath
for about 30-40 second and cut...beautiful, artifact free, duplicable and
fast.

What are the reasons s/he doesn't want the more standard snap freeze
methods?  Is it a time or expense issue?

Cheryl



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gudrun Lang
Sent: Wednesday, November 21, 2007 11:51 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] frozens from lung-tissue

Hi to all,

I need the help of those, who have experience on making good frozens from
lung-tissue. We look for a method for freezing lung-tissue without the usage
of liquid nitrogen, co2 or isopentan. - Just on the cryocut. The head of the
institute is not satisfied with the quality and wants us to do better. Our
usual method is to make a OCT-layer on a chuck, let it freeze, give the
tissue on it, surround with OCT, put the cold weight on it, let it freeze
and make the sections.

We tried putting the tissue in the liquid OCT on the chuck without a layer
and  freezing it in a mold with the chuck on top. I also tried first
freezing the piece solely in liquid nitrogen or isopentan, then putting it
on a chuck. All in all - my boss wasn't happy with it.

 

Any input will be appreciated. Thank you in advance

Gudrun Lang