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--></style><title>Re: Sucrose as a
cryoprotectant</title></head><body>
<blockquote type="cite" cite>Donna,<br>
I hope that you don't mind a quick question...<br>
I've had to do frozens on tissue that was cryoprotected with sucrose
then snap frozen and brought to me to cut. I had numerous problems
with a rim of sucrose that was not frozen and sort of sticky around
the tissue while the tissue and the OCT was frozen. Is this normal or
did the investigator's lab screw up when freezing the tissue (one was
mouse brain and one was mouse embryo).<br>
Do you have a procedure for freezing cryoprotected tissue and for
cutting them?<br>
Thank you!!<br>
Andi Grantham<br>
.........................................<span
></span>............................<br>
: Andrea Grantham, HT(ASCP) Dept. of Cell
Biology & Anatomy :<br>
: Sr. Research Specialist
University of
Arizona <span
></span> :<br>
: (office: AHSC
4212) P.O. Box
245044 <span
></span
>
:<br>
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:........................................<span
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http://www.cba.arizona.edu/histology-lab<span
></span>.html</blockquote>
<div><br></div>
<div>------------------</div>
<div><br></div>
<div>Andrea, Here are some musings. I hope they
help. -Donna</div>
<div><br></div>
<blockquote type="cite" cite>problems with a rim of sucrose that was
not frozen and sort of sticky around the tissue while the tissue and
the OCT was frozen.</blockquote>
<div><br></div>
<div>Optimal freezing temps for OCT, the tissue, and the sucrose
solution used could well be<u> different</u>!</div>
<div><br></div>
<blockquote type="cite" cite>Is this normal or did the investigator's
lab screw up when freezing the tissue (one was mouse brain and one
was mouse embryo).</blockquote>
<div><br></div>
<div>I doubt its normal or desirable, but won't hazard a guess as to
a specific screw-up...</div>
<div><br></div>
<blockquote type="cite" cite>Do you have a procedure for freezing
cryoprotected tissue and for cutting them?</blockquote>
<div><br></div>
<div>I freeze fixed, cryoprotected rat brain by itself, or in a
"block" of the Sucrose cryoprotectant solution. It
can be frozen rather slowly, as I've mentioned before. We
typically just surround it with a heap of pulverized dry ice and cut
15-30micron sections on a freezing stage of a sliding
microtome. Be sure to leave it long enough to freeze
completely through before you try sectioning. </div>
<div>When using a cryostat, it is crucial to find the correct cutting
temperature for your preparations! Fixed brain needs to be a
bit colder [-20 to -22deg.C] than unfixed liquid Nitrogen snap-frozen
brain [-16 to -19deg.C], for example. OCT gets 'gummy' when in
contact with formalin, so use care to rinse the tissue in
sucrose/buffer before freezing in OCT. Whether or not you want
a surrounding matrix of some sort probably depends on how thick your
sections will be, and whether you want to go back to the block to cut
more sections later. Directly frozen tissue might cut well, but
tends to dessicate after a fairly short time in the cryostat, as you
may know if you need to cut step sections throughout an entire
brain...</div>
<div><br></div>
<div>I freeze cryoprotected, fixed embryos [rat -- all embryonic
ages, and up to P-2] in a 50:50 mixture of Shandon Immumount [yes!]
and OCT in a plastic embedding mold [peel-away]. I use a
slurry of crushed dry ice in absolute ethanol to conduct the cold
from the bottom of the tissue mold while I visualize it from
above. You must rinse the fixative off rather well with
cryoprotectant in buffer [I use 20% for rat embryos], else it will
CONGEAL the OCT mixture! I rinse the tissue in one
change of the mixture and freeze in a fresh solution. This
makes positioning the embryo easier during the freezing
process. Initial correct positioning is usually important, as
the block becomes opaque upon freezing. Note, this is not
embedding in the sense used for paraffin, but merely supportive
encapsulation as with the usual OCT (however, its very much
better!).</div>
<div><br></div>
<div>The Immumount essentially acts as a partial antifreeze for the
OCT, resulting in a matrix that will be harder than OCT and will thus
better support fragile embryonic tissues. Of course it must be
frozen and cut at a much lower temperature than usually used for
brain: I use -26deg. C. or lower. If your cryostat won't
hold that low a temperature, you might try experimenting with a
different proportion of OCT and Immumount to form a solid, cuttable
block. It will, of course, be softer and may not preserve
the embryo morphology as well. I used this method in the
paper, Simmons, et al, Genes and Development 4:695-711;
1990.</div>
<div><br></div>
<div>The original idea for the 50:50 mixture is from a paper by Jules
Elias and colleagues: Jones, Elias and Schecter, "An
Improved Method for Embedding Retina for Cryosectioning", J.
Histotechnology/ vol 9, no 3/ September 1986. The
original product they used in combination with OCT was Lerner Labs'
Aqua-Mount, whose formulation was later sold to Shandon.</div>
<div><br></div>
<div><br></div>
<div>-- <br>
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></span>=+=+=+=+=+=+=+=+=<br>
Donna M. Simmons
HTL(ASCP),<x-tab> </x-tab> PhC
(Neuroscience Graduate Program)<br>
Research Associate, Department of Biological Sciences<br>
University of Southern California<br>
Hedco Neurosciences Building <x-tab>
</x-tab><x-tab>
</x-tab>voice: 213/740-3166<br>
3641 Watt Way - Room428<br>
Los Angeles, CA 90089-2520<x-tab>
</x-tab><x-tab>
</x-tab>fax: 213/741-0561<br>
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webpage: <http://www-hbp.usc.edu/people/donna.htm<span
></span>> </div>
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