Re: Panc. islet B cells, ald.fuchs. & more
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | jennifer.hoover@pharma.Novartis.com |
Reply-To: | |
Date: | Sat, 3 Jul 1999 23:05:48 -0400 (EDT) |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
On Fri, 2 Jul 1999 jennifer.hoover@pharma.Novartis.com asked
for advice about an old and still poorly understood method:
> under the renal capsule. I have consulted Sheehan's book for Gomori's Aldehyde
> Fucshin staining of pancreatic islet cells but have had little success. The
> stain functions beautifully for the elastin tissue in the vasculature but there
> is no staining of islet cells; even in normal pancreas tissue.
There's quite a lot of literature on this subject, most of it
15-35 years old and therefore rarely read. Assuming that your
aldehyde-fuchsine solution works (and you say it's staining
elastin OK) it should be able to stain pancreatic B cells, almost
certainly by virtue of their content of insulin. The first thing
you do is oxidize the -S-S- linkages of this cystine-rich protein
to cysteic acid, which is a sulphonic acid and can bind
aldehyde-fuchsine strongly.
The easiest way to do this is to treat hydrated paraffin sections
with 0.5% potassium permanganate in 2% sulphuric acid for 5 minutes,
then put them in 1% aqueous oxalic acid for about 30 seconds, until
the brown colour (manganese dioxide) has gone. Cystine-rich proteins
(including insulin, keratin and classical neurosecretory
material or neurophysin) are now bristling with strongly acidic
sulphonate anions. Any basic dye will cling to them, including
alcian blue at pH < 1, chrome-haematoxylin, or aldehyde-fuchsine.
Aldehyde-fuchsine is more clinging than many other coloured
cations, for reasons that haven't been fully sorted out. It resists
extraction by acid-alcohol and may be covalently bound, perhaps
to parts of the insulin (or whatever) molecule near the oxidized
cystines.
A strange and unexplained property of aldehyde-fuchsine is its ability
to stain pancreatic B cells that have not been treated with a strong
oxidizing agent. If you omit the oxidation the age and method of
preparation of the aldehyde-fuchsine solution are critical. An
important (for those who care) paper is Mowry,RW 1978. Aldehyde
fuchsin staining, direct or after oxidation: problems and remedies
with special reference to human pancreatic B cells, pituitaries and
elastic fibers. Stain Technol. 53: 141-154.
An aldehyde-fuchsine solution that stains elastin will not
necessarily stain pancreatic islet B cells without prior
oxidation. Another important point is that you must use
pararosaniline as the "basic fuchsine" when you prepare an
aldehyde-fuchsine reagent. Certified pararosaniline is sold by
the major American vendors. A batch of b.f. not identified as
pararosaniline will not work in any aldehyde-fuchsine technique.
There is a lot more that could be said.
If any readers of this HistoNet reply want more information
in the way of references etc, please email me without
involving the whole group.
My email address is KIERNAN@UWO.CA
It is remarkable that Gomori, a founding father of
histochemistry, should have his name attached to this
inadequately understood method for B cells when he developed
techniques of enzyme histochemistry that have changed only
in detail since the late 1930s.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
E-mail: kiernan@uwo.ca
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