RE: [Histonet] Thioflavin S staining

From:"Leskovjan, Andreana"



Hi John,

Thank you very much for your reply.  Unfortunately, the experimental
design does not allow fixation nor cryoprotection.  Also, the tissues
look very good before staining and also when staining on regular glass
slides.  Do you have any advice on keeping the tissues intact during
staining on substrates other than glass?

 

Thanks,

Andreana

 

________________________________

From: John Kiernan [mailto:jkiernan@uwo.ca] 
Sent: Tuesday, April 22, 2008 2:47 PM
To: Leskovjan, Andreana
Cc: histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Thioflavin S staining

 

Your description gives the impression of damage caused by slow freezing
without cryoprotection. Ice crystals form in the tissue, leaving behind
holes. Prevention: adequate fixation in buffered formaldehyde, followed
by cryoprotection (usually soaking in 30% aqueous sucrose for a day or
two; until the brain sinks). Even with cryoprotection, the specimen
should be frozen as quickly as possible. There have been many Histonet
postings on this subject over the years. They can be found at
www.histosearch.com  .
 
John Kiernan
Anatomy, UWO
London, Canada.
= = =
----- Original Message -----
From: "Leskovjan, Andreana" 
Date: Tuesday, April 22, 2008 12:16
Subject: [Histonet] Thioflavin S staining
To: histonet@lists.utsouthwestern.edu

> Hi,
> 
>  
> 
> I need to stain 30 um frozen, unfixed mouse brain sections with
> Thioflavin S.  The sections are mounted onto a very thin 
> plastic film
> called Ultralene.  I know it's not the best but necessary 
> for our
> measurements.  I have been using a very simple procedure so 
> far with
> minimal chemicals to avoid interference with the 
> measurements:  50%
> ethanol for 5 min; 0.0125% Thioflavin S for 3 min; wash in 50% 
> ethanol,then nanopure water.  The staining works however 
> the tissue is almost
> destroyed.  There are holes, tears, and folds everywhere, 
> which makes
> the plaques difficult to see and measure.  I've tried many 
> combinationsof the above and this is the best I could get.  
> Does anyone have a
> suggestion on how to prevent this and get better looking 
> sections?  
> 
>  
> 
> Thanks,
> 
> Andreana Leskovjan
> 
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