[Histonet] Will brain tissue shrink even more ifstoredin70%alcoholprior to processing?

[Patsy Ruegg]

For IHC it is usually recommended that tissues be optimally fixed in NBF
then transferred to 70% alcohol for storage before processing to limit the
ill effects of cross linking of proteins by over fixation with aldehyde
fixatives.  If buffer salt crystals can form using these methods we we
reconsider this?  I have always been a believer in washing after fixation
but most of us do not have the luxury of having the time to do so.  As I
researched a presentation I was doing on IHC I ran across this: "A little
known fact is that if tissues were washed in running tap h202 (10-20 min.)
after aldehyde fixation most epitope retrieval procedures would be
unnecessary."  Now there may be another reason to wash after aldehyde
fixation????
Patsy 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Vinnie Della
Speranza
Sent: Wednesday, June 01, 2005 8:18 AM
To: jkiernan <@t> uwo.ca
Cc: jchladny <@t> cvm.uiuc.edu; histonet <@t> lists.utsouthwestern.edu;
clarissabush <@t> sbcglobal.net
Subject: Re: [Histonet] Will brain tissue shrink even more
ifstoredin70%alcoholprior to processing?

I hadn't considered the possibility of buffer salt crystals forming the
vacuoles observed by the author and I don't know if she is on this list and
would care to comment but this is an interesting premise worth
investigating. I would think that a thorough water wash prior to going into
alcohol would help to clear this up.
thanks for the feedback.
Vinnie

Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974

>>> John Kiernan <jkiernan <@t> uwo.ca> 05/27/05 04:27PM >>>
In the HistoLogic May 2000 paper the duration of the initial formaldehyde
fixation is not stated; only that the brains were "previously well fixed in
neutral buffered formalin." Frequently specimens are not fixed for long
enough to make them resistant to bad effects of solvents etc. 

It's also seems from the paper that the test pieces of brain were passed
directly from the buffered fixative into 70% alcohol. This causes
precipitation of sodium phosphate in the tissue (see Freida Carson
1996 "Histotechnology" 2nd edn, p.27). The control specimens in the
HistoLogic paper were passed from buffered formalin to 60% ethanol (which
safely extracts the phosphate buffer salts). Could the holes in the white
matter be made by buffer salt crystals rather than forming slowly during
storage in 70% ethanol?

Just a thought!
--
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/ 
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________
Vinnie Della Speranza wrote:
> 
> You may wish to look at an article authored by Jane Chladny, published in
HistoLogic, May 2000, entitled "Artifact in Tissues Held in 70% Ethanol"
> 
> the author reported the appearance of 'vacuole' artifact in nervous
tissues observed in a variety of mammalian tissues after storage in 70%
ethanol.
> 
> you can access the article by selecting the link for HistoLogic at 
> www.sakuraus.com
> 
> CM Bush wrote:
> >
> > Dear Histonet,
> >
> > Hello, here is my first post to the list, thank you in advance for your
help.
> >
> > Summary:
> > Having problems with mouse and human brain tissue shrinkage durning
processing, but would like to better preserve antigens, concerned storage of
tissue 70% ethanol will shrink tissue even further:
> >
> > We have lots of brain specimens, stored in formalin for a long time
(months up to 10 years), both human and mouse.  We perform
immunohistochemistry on paraffin embedded brain.  Of course, there is the
massive over fixation. Also the tissue shrinks a lot during processing.
> >
> > In my last lab, after brain tissue had been fixed in formalin for 7 days
we would store the tissue in 70% ethanol.  I'd like to start storing the
brain tissue I work with now in 70%, but I'm worried about the tissue
possibly shinking too much, as the tissue already seems to shrink by greater
than 60% after processing.
> >
> > (Previously, we would gradually increase the alcohols, 30% for 1 
> > hour, then 50% for an hour in a bucket, room temperature on a rocker 
> > table, then put the cassettes into a bucket of 70% and store at room 
> > temp or 4C- does this process help any with controling the shrinkage 
> > factor?)
> >
> > Maybe this is a little bit long...thank you very much for your time.
> >
> > CM Bush
> ---------
> 
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> 
> Vinnie Della Speranza
> Manager for Anatomic Pathology Services Medical University of South 
> Carolina
> 165 Ashley Avenue  Suite 309
> Charleston, SC 29425
> Ph: 843-792-6353
> fax: 843-792-8974
> 
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