PFAAB, PAS, Orange G revisited

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From:Sarah Christo <> (by way of histonet)
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Dear John,
   Thank you for the reply.  I'm making solutions and have two questions.
Can the acidified potassium permanganate be made up as a stock solution
with the acid or should the acid be added just before use?
  Also, just as a curiosity, (since there has been a discussion going on
Alcian Blue), I have a variance in Alcian blue solutions made.  I have many
shelves of old dyes and picked what I thought was the newest dye to make up
the Alcian Blue pH 1.2.  This was a Sigma dye from 1985.  It formed a
precipitate so I filtered it and it is a pale blue, weak looking solution.
I then tried a Chroma Alcianblau 8GS from 1971, and this did not
precipitate like the other (only slightly) and is filtering to make a much
stronger looking darker blue solution.  These two dyes in powdered form
also look different.  The Sigma is a finer powder, darker blue, the Chroma
is a more granular consistency and a lighter blue.  Any thoughts on which
one of these would be better to use?  I plan to run a control through on
both of these first of course, but was curious about the differences.
Thanks, Sarah

Sarah Christo, HT (ASCP)
Texas A&M University
College of Veterinary Medicine
Dept. of Vet. Anatomy & Public Health
Histology Laboratory
College Station, TX  77868-4458
phone (409) 845-3177
fax (409) 847-8981

>>> "J. A. Kiernan" <> 11/05 10:04 AM >>>
On Thu, 4 Nov 1999, Sarah Christo wrote:

> I was wondering how hazardous and what precautions need to be made in
>using Performic Acid.  This technique mixes formic acid with hydrogen
>peroxide and sulfuric acid.  It says to keep the temperature below 25
>degrees C.  It also decomposes in a few hours.  Peracetic Acid (glacial
>acetic acid, hydrogen peroxide and sulfuric acid) can be substituted,
>would this be any safer???

  Performic acid isn't for the faint hearted. It's very
  corrosive and there is a potential explosive hazard if
  it comes into contact with metals or their salts. A
  major difficulty in using it is that it continuously
  generates tiny bubbles of oxygen, and these seem to
  accumulate abundantly between slide and sections,
  causing detachment and other damage.

  Peracetic acid is more stable, and can also be made
  easily in the lab. A more easily used oxidizing agent
  for the staining method in the header of your email
  (presumably for pituitary cell types and neurosecretory
  material) is acidified potassium permanganate (Pasteels,JL
  & Herlant,M 1962. Notions nouvelles sur la cytologie de
  l'antehypophyse chez le rat. Z. Zellforsch. 56: 2-39).

  Put the hydrated slides in 0.5% KMnO4 in 2% H2SO4 for
  5 minutes, then in 1% oxalic acid (H2C2O4.2H2O) for
  about 30 seconds, to remove the brown MnO2 deposit from
  the sections.

  Like performic or peracetic acid, the acidified permanganate
  oxidizes cystine to cysteic acid, which is strong (a
  sulphonic acid) and therefore able to bind cationic dyes
  at pH 1 or lower. Cystine-rich proteins (such as
  neurophysin, TSH and keratin) are therefore stainable
  by acidic solutions of alcian blue (or any other basic
  dye), chromium-haematoxylin, aldehyde-fuchsine etc, after

  For anterior pituitary cell-types you need quite thin
  sections (4 micron), but thicker ones (10-12 micron) are
  better if you want to admire the neurosecretory perikarya
  in the hypothalamus. Zenker, Helly and Heidenhain's SUSA
  are good fixatives for both purposes. Good luck!

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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