Hello,


	Thanks to all who replied to my question about the freezing
artifact in large avian brains that was sent by Laurie Reilly.  Suggestions
to eliminate the freezing artifact came basically in two categories,
cryoprotectant and freezing methods.  All who mentioned croyprotectants
agreed that infiltrating with 25-30% sucrose rather than 20% sucrose would
decrease the freezing artifact.  If time is not of the essence, a graded
sucrose infiltration starting with 10% and moving gradually to 20% and then
30% over a few days was recommended.  To combat the stickiness of 30%
sucrose it was recommended that, before freezing, the brain is coated in
PBS and then OCT cut 50% with buffer.
	It was suggested that liquid nitrogen by itself is too cold for
freezing brain tissue and that this typically will cause the cracks that I
observed.  Four individuals suggested using liquid nitrogen in combination
with other agents.  One individual recommended cooling isopentane in liquid
nitrogen and immersing the sample for 12 seconds.  Two other researches
suggested using this same method but instead of immersing the sample
placing it on a metal chuck on top of the isopentane and allowing the
freezing to progress up the tissue.  This alternative was suggested to
further reduce the occurrence of cracks in the tissue.  Hexane as an
alternative to isopentane was also recommended as a way to increase the
freezing temperature and avoid cracking.  One researcher recommended using
the isopentane method in combination with surrounding the brain with
crushed dry ice to speed the freezing front.
	About half of those responding said they had found that using
crushed dry ice, rather than pellets of dry ice, provides a satisfactory
method of freezing.  The dry ice was used either by itself or in
combination with a cold plate or a "bunker" of larger pieces of dry ice
placed around the brain.
	The methods discussed in the recommended reading "Crotechniques for
light microscopy" by Mark Donovan and Henry Preston
(http://home.primus.com.au/royellis/fr.htm) were also very useful.
	I tested several of the suggested techniques alone and in
combination.  I found that immersion in chilled isopentane caused some
cracking while allowing a freezing front to progress up the tissue did not.
Freezing with crushed dry ice alone did not eliminate the freezing
artifact, although it did reduce it. Infiltration with 30% sucrose compared
to 20% sucrose reduces freezing artifacts.
	I settled on the following techniques to eliminate the freezing
artifact: Place brain in 20% sucrose at 4C until sinks (2 days).  Place in
fresh 20% sucrose for (2 days).  Hemisect brain and place the right half in
100 ml of 30% sucrose, 5% DMS0, PBS at 4C for 3 days. (One half of brain
was treated with previous methods for comparison with older samples that
have the freezing artifact).
	For freezing, chill isopentane using a dry ice acetone slurry.  Use
a volume of isopentane that will come just up the bottom edge of the
specimen disk on your chuck.  I found 25ml in a 50 ml beaker to be about
right.  Place a metal chuck on dry ice and "glue" the brain on in the
proper orientation. Use a syringe to lightly coat the brain with OCT cut
50% with PBS. Fit a cardboard ring around the brain on the chuck and place
the chuck on a coat hanger holder.   Lower the chuck on to the surface of
the isopentane and spoon crushed dry ice into the cardboard ring.  I kept
the tip of the brain clear of dry ice on my first attempt with these
methods so I could watch the freezing front progress.  The tissue took
about 1 minute 30 seconds to freeze. The brains were then cut in the
cryostat at 40u and about -20C.  This eliminated the freezing artifact.

Thank you,

Lainy

Lainy Day
Dept. of Zoology and Tropical Ecology
James Cook University
Townsville, QLD 4811 Australia
Office: 61+ 7 4781 6893
Lab: 61+ 7 4781 4601
FAX: 61+ 7 4725 1570