[Histonet] cryoprotection possible?

[Merced Leiker]

I agree with Frances and anh2006. Cryospreservation is important for brains 
especially and must be done after fixation, before freezing, and the 
glutaraldeyde can irreversibly mask antigens.

Merced

--On Wednesday, March 18, 2009 8:49 AM -0500 "Swain, Frances L" 
<SwainFrancesL <@t> uams.edu> wrote:

> 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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Merced M Leiker
Research Technician II
354 Biomedical Research Building
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
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Ph: (716) 829-6033
Fx: (716) 829-2725

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