[Histonet] sucrose infiltrated muscle

[Due, Brice]

Steve, You didn't mention what the problems have been, nor which "snap" freeze
protocol you've been using. But even whole adult rat quads should not be too big
to snap freeze. Nor whole limbs. I've frozen entire rat pups without artifact.
Human bxs up to 2.0x2.0x3.0cm without freeze atrifacts (those were autopsy
steaks).
 
Are you using LN2 chilled isopentane? If so, try letting the isopentane solidify
or almost solidify. Then remove the isopentane in the metal beaker and break it
up as it thaws. You only get about 2min to work depending on how much isopentane
you are using. I get the best (quickest) freezes when the isopentane is a chunky
slush. The solid bits act as a heatsink so the liquid can stay at freezing point
longer. When you dunk the tissue be careful about bumping the chunks during the
first 0.5-1.5sec, then agitate agitate agitate. If you don't agitate, the
isopentane immediately surrounding the tissue will have warmed up and do you no
good. Near freezing pt, the liquid is too thick to convect well. So agitate. 
 
Time to freeze all the way to center is never certain, so I always spend
20-30sec even for small bxs. Once all the isopentane chunks are gone is one way
of timing the freeze. This can cause the tissue to crack, but you can either
learn to cut cracked tissue or repair the cracks with cold OCT (refrigerator
temp or lower). You can't repair freeze artifacts once they happen, so choose
your trade-off.
 
Are you sure your freeze artifacts (if that is what you are getting) are
happening at the time of snap freeze? IF the tissue ever thaws partially then
ice crystals will grow as it re-freezes. Check all your handling to eliminate
any possibility of thaw. Especially: how do you mount the tissue for sectioning?
Do you use warm room temp OCT? Any source of heat can cause problems, even long
after the snap freeze.
 
Good luck,
-brice
 


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-----Original Message-----
From: Steven Coakley [mailto:sjchtascp <@t> yahoo.com]
Sent: Monday, August 22, 2005 1:30 PM
To: Due, Brice
Subject: RE: [Histonet] sucrose infiltrated muscle


Rat/mouse leg muscle for research.  Often a "tad" to big for good snap frozens.

"Due, Brice" <BDUE <@t> PARTNERS.ORG> wrote: 

What kind of "inconsistencies" are you experiencing? Are these clinical bxs?

-brice
Neuropathology
Brigham & Women's Hosp.
Boston


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OF ANY ACTION IN RELIANCE UPON, THIS INFORMATION BY PERSONS OR ENTITIES OTHER
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu]On Behalf Of Steven
Coakley
Sent: Monday, August 22, 2005 12:21 PM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] sucrose infiltrated muscle


Has anyone ever worked with fixed muscle infiltrated in sucrose then snap
frozen. I'm looking at the option due to inconsistencies with regular snap
frozen muscle?

Steve


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