[Histonet] cryoprotection possible?

[Swain, Frances L]

Usually the cryoprotection is carried out after the specimens are fixed and before they are frozen.  If you have a sample you can spare you might try making up some 20% Sucrose placing the frozen sample in it. putting it in the refrigerator and letting it stay in the 20% sucrose until it drops to the bottom of the container.  You might have to change the 20% Sucrose a couple of times as you know sucrose can grow bacteria easily.  When the specimen has dropped to the bottom of the container, remove it, rinse in a couple of changes of distilled water and quick freeze the sample.  Try cutting and staining the sample and see if the morphology is good if it is then you can do all of your samples to correct the problem.  There should be no thawing between removal of the sample from the storage to the cryostat. 
Hope this helps
Frances Swain

________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Dr. med. Frauke Neff [nefff <@t> staff.uni-marburg.de]
Sent: Wednesday, March 18, 2009 3:47 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] cryoprotection possible?

Dear Histonetters,
I'm supposed to do a noradrenaline ihc on rat brains that have been snap frozen
and afterwards glutaraldehyd fixed. While the morphology is okay in the
"test"-tissue we used to establish the protocol, it was very poor in the tissue
of the trial animals.
It appeared mooth eaten and disrupted.

I assume the brains were thawed and refrosted due to moving the samples between
several cities. Does anyone know a method /a tip how I can save some proper
morphology?

I checked the archive and found sucrose as cryoprotectant, but this is supposed
to do it while the tissue is frozen or is it possible to use it while the
tissue thaws?

Thank you all in advance for your patience and help,


Frauke


--
Dr. med. Frauke Neff
AG Neurologische Therapieforschung
Neurologie
Philipps-Universität Marburg
Rudolph-Bultmann Str. 8
35039 Marburg
06421/5866304





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