[Histonet] frozen sections

From:"Emerson, Rachael"

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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