Re: [Histonet] mouse heart morphology
Sorry buy -40C is NOT cold enough for freezing our murine tissues. I
believe that others have experienced this problem also, the one of getting
freezing artifact/damaged morphology with inside cryostat temperature
Nothing scary about liquid nitrogen usage, although Isopentane is more of a
worry for storage. I was thinking more of precooling the metal device
containing wells i.e surround the device with liquid nitrogen, then you
have the wells at the extremely cold temperatures. Liquid nitrogen in the
wells would not be ideal, to be sure.
We do this with a metal block sitting in liquid nitrogen so the metal
block is at liquid nitrogen temperatures, then place an OCT embedded murine
tissue (inside a Tissue Tek plastic mold) on top of the very cold metal
block. This permits murine tissue to freeze at a much colder temperature
and without artifact.
I have seen your device and like its design, but if mouse tissue cannot be
snap frozen at colder temperature than -40C, then we can't use it due to
terrible morphology. Cryostat freezing temperatures for murine
cryomicrotomy simply do not do the job well enough. It is the water ice
crystal formation inside the tissue that presents the big problem and the
colder the freezing temperature, the smaller the ice crystal formation -
hence snap freezing in the true sense of the words. Precooling OCT and a
tissue is not feasible when you have 10 mice lined up and collecting 15
tissues out of each mouse as fast as you can dissect and snap freeze plus
the actual snap freezing takes only seconds or so - extremely fast.
I would love to try your device in the way we use our metal block
surround by liquid nitrogen as your device has a very efficient
design. There is no doubt it works very well for clinical laboratories
for rapid diagnostic work, but for our fussy murine tissues, colder
temperatures must prevail.
There is an excellent discussion by Charles Scouten on snap freezing
biological samples can be found at www.myneurolab.com with details on ice
crystal formation, etc. - what we experience with murine tissue.
At 10:52 AM 3/7/2005, you wrote:
>I have not experimented with liquid nitrogen in my wells. Sounds scary!
>In order to reduce freeze artifact using my system I have a few suggestions.
>1) Cool the tissue and OCT to refriderator temp. I think this is a smart
>idea for any
> snap freezing technique. Why should we add the calories to go from room
> temp to
>0 degrees C, when it takes a lot less calories to go from 1 degree to O
>2) My bars can be cooled down as low as about - 40 C and still maintain
>the adhesive quality of the cold metal which is what allows us to be most
>precise. Chucks can be put in
>liquid nitrogen to get them super cooled.
>I would love to see someone try this and tell me how their result came
>out. I think it would freeze pretty quickly.
>Any takers? Got to run for a frozen.
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)
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