[Histonet] cryoprotection possible?

Wed Mar 18 08:57:18 CDT 2009

What I do is take the specimen out of 30% sucrose, blot and place in OCT for maybe 30-60 minutes, then place in fresh OCT and freeze.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Swain, Frances L
Sent: Wednesday, March 18, 2009 8:50 AM
To: anh2006 <@t> med.cornell.edu; Dr. med. Frauke Neff; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] cryoprotection possible?

You are so correct, I forgot you do not rinse the sucrose out you blot it and then freeze.  Sorry about that. I do not cut frozen sections very often and I forget.

Frances L. Swain HT(ASCP) A. A. S.
Special Procedures Technician
Department of Orthopaedic Surgery
Center for Orthopaedic Research
Barton Research Building 2R28
4301 West Markham Street
Little Rock AR 72205
(501) 686-8739 PHONE
(501) 686-8987 FAX
swainfrancesl <@t> uams.edu email
-----Original Message-----
From: anh2006 <@t> med.cornell.edu [mailto:anh2006 <@t> med.cornell.edu]
Sent: Wednesday, March 18, 2009 8:41 AM
To: Swain, Frances L; Dr. med. Frauke Neff; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] cryoprotection possible?

Good advice indeed. However, I don't recommend you rinse in water after sucrose. Sort of defeats the purpose. Instead if you need to remove excess sucrose rinse in either 50/50 sucrose/OCT or just OCT.

Also, glutaraldehyde fixation usually renders tissue difficult to immunostain. You have to consider whether your fixation is appropriate for your antigen/antibody.

-----Original Message-----
From: "Swain, Frances L" <SwainFrancesL <@t> uams.edu>

Date: Wed, 18 Mar 2009 08:12:45
To: Dr. med. Frauke Neff<nefff <@t> staff.uni-marburg.de>; histonet <@t> lists.utsouthwestern.edu<histonet <@t> lists.utsouthwestern.edu>
Subject: RE: [Histonet] cryoprotection possible?

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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