Re: Panc. islet B cells, ald.fuchs. & more

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From:"J. A. Kiernan" <>
Date:Sat, 3 Jul 1999 23:05:48 -0400 (EDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

On Fri, 2 Jul 1999 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

  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

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