Re: [Histonet] Will brain tissue shrink even more if storedin70%alcoholprior to processing?
|From:||"Vinnie Della Speranza" |
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 Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue Suite 309
Charleston, SC 29425
>>> John Kiernan 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
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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