[Histonet] frozen sections

[Kinsley, David]

Hi Rachael,

I use OCT embedding compound supplied by Sakura/Tissue Tek.  I put a small
amount in the bottom of a plastic embedding mold, orientate the specimen,
fill the mold with OCT and then immerse the entire mold into a slurry of
2-methyl Butane (isopentane) in dry ice.  With the technique you can keep
the sample immersed a few minutes (2-3)without it cracking.  It is best to
immerse the sample, as it will freeze from all sides rather than from the
bottom up as a floating technique would do.  Liquid Nitrogen freezes
quickly, but you have to be very careful as the blocks tend to crack very
often.  Freezing in the -80 is too slow and ice crystal will form in your
samples.  Good luck



Dave


-----Original Message-----
From: Emerson, Rachael [mailto:Rachael_Emerson <@t> URMC.Rochester.edu] 
Sent: Friday, May 27, 2005 3:51 PM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] frozen sections


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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