[Histonet] cryoprotection possible?

[nefff <@t> staff.uni-marburg.de]

We tried nearly everything for the noradreanline IHC and did't get any  
staining. After several calls with the techs of the company, they told  
me I've to do a glutaraldehyd fixation, every other fixation will wash  
the noradrenaline out of the tissue (even the cryosection did't give a  
stain).
Using adrenal glands as positive control, I got a weak positive stain  
using the GA-Fixation. But I'm not happy with this, because the  
samples I got are FFPE or snap frozen. So what to do to proceed?  
Because I don't know any way back through the formalin fixation, I  
decided to go on with the snap frozen tissue.

I would be very happy, if someone would tell me: "Hey, thats all  
bullshit, you just need the antibody XXX of the company YYY and  
verything will work fine with this noradrenaline IHC"

Until this happens, I try with the snap-frozen material, keep my  
fingers cross, throw pennies in every fontaine, rub oil-lamps and lion  
noses....

You see, I'm desperately needing help,

Frauke


Zitat von anh2006 <@t> med.cornell.edu:

> 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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