Not seeing any replies to this, I thougt I would chip in my 2 cents worth ......

Laurie Reilly wrote:

> Dear All,
> Could anyone please help with this query from one of our researchers:-
>
> I am trying to determine the best way to fix a freezing artifact.  The
> brains have been perfused and post fixed for a 4 weeks or more.  They have
> then been placed in 20% sucrose in .1M phosphate buffer until they
> sink.

    After the brain sinks, change to fresh 25 or 30% sucrose and wait until it
sinks in that solution. Some use glycerin in the sucrose too. Don't remember how
much.

> The brains were then frozen by  burying them under shaved dry
> ice.  Sections are to be cut in the cryostat at 40 um to preserve
> neuroarchitecture.
>          I am almost positive the distortion in my cut and stained sections
> is due to the freezing artifact.

    In my experience, freezing artifact is usually manifested as holes in the
tissue. "Swiss cheese" if you will. The slower the freezing, the bigger the
holes. What sort of "distortion" do you see?

> The artifact is less severe in smaller
> brains, and is worse on samples that were frozen with dry ice pellets
> rather than shaved dry ice.  Most significantly, I froze one sample by
> placing it above liquid nitrogen and the artifact disappeared.  However,
> the brain broke into many pieces.

    Probably too cold. Cool some 2-methyl butane ("isopentane") in liquid
nitrogen until it is thick and slushy. The brain is "glued"  (OTC or whatever) to
a metal chuck or object disk (MUST be something that conducts heat/cold quickly)
and the metal chuck is put into the 2-methyl butane. You can watch the "freezing
front" progress up the tissue. If at the same time you could surround the brain
with crushed dry ice, even better. I make a cylinder out of heavy paper to fit
over the brain. The pour crushed dry ice into the cylinder.

>          The main problem I am having is that my specimen are large avian
> brains (approximately 20 mm from anterior to posterior, 25 mm at the widest
> point of the telencephalic hemispheres, and 15 mm from dorsal to
> ventral).  I could cut the brains in half from anterior to posterior but
> the brain would will still be 12.5mm as the smallest dimension.  I am
> hesitant to make coronal cuts for a couple of reasons; I want to preserve a
> measurement of whole brain and would lose some sections on alignment of
> each specimen piece in the cryostat, the architecture of the brain has not
> been mapped so trying to make the cuts in the least important areas would
> be guess work.  I would like to examine 4 non-localized brain regions as
> well as preserve the architecture so that other researchers could examine
> their own regions of interest.

    When I was a grad student one of my profs did whole squirrel brains, which
were larger than what you are working with. He had no problems but he froze from
the chuck and surrounded the brain with dry ice.

Geoff
--
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029
mcauliff@umdnj.edu
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