[Histonet] frozen sections

[Vinnie Della Speranza]

There are two very good articles discussing preparing tissues for frozen cryotomy  by Gayle Callis in the Spring and Autumn 2004 issues of HistoLogic.
you can access them by using the HistoLogic link at
www.sakuraus.com





Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974

>>> "Emerson, Rachael" <Rachael_Emerson <@t> URMC.Rochester.edu> 05/27/05 03:51PM >>>
Hello. I am in need of some help with trying to freeze mouse embryos for
frozen sectioning.
I am dissecting mouse embryos out in PBS (Embryonic day 8.5-14.5) and
freezing them in VWR's Neg 50 Embedding Media. 

Initially I put a small amount of Neg 50 in the bottom of a Polyscience
Plastic Peel-Away Mold, oriented the embryo, and then added more Neg 50.  I
then put the molds in the -80 Freezer and let them solidify.  They seem to
cut OK, but the morphology was terrible.  Not so much in the E14.5, but
other embryos looked very degraded.

Next, I tried the same procedure but froze them by floating the mold in
ethanol with dry ice. Still the morphology was terrible. I tried to freeze
the embryos directly and then embed them, but they just turned to mush.

On my final attempt I repeated the same procedure, but froze the mold by
holding the bottom in liquid nitrogen. The block froze in seconds, but when
I took them it out of the mold it was cracked and very hard to cut.  The few
sections I did manage were terrible and you could see the ice crystals in
the embryos.

I would really appreciate any advice or suggestions you have to offer.
Thank you
Rachael Emerson




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