Re: sialic acid procedure

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From:"J. A. Kiernan" <> (by way of Marvin Hanna)
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On Wed, 26 Jan 2000, Jill Songer wrote:

> Boss has asked me to find a procedure for sialic acid in basement
> membranes. Can anyone out there help?? Many thanks.

  Your boss is getting into quite deep water here, and needs
  to get out a histochemistry textbook, read the chapter on
  carbohydrate histochemistry, and consult the references that
  seem most relevant to questions he is trying to answer.

  Sialic acid is a general term for a family of 9-carbon
  sugars that have 5 carbons (C2-C6) and an oxygen as part
  of a hexose-like ring, and 3 carbons (C7, C8 and C9) as a
  side-chain. C1 is the carbon of a carboxylic acid group,
  which is a side-chain attached to C2. There is also an
  N-acyl substituent on C5. These sugars are (hope I'm not
  out of date) produced only by animals and are incorporated
  into oligo- and polysaccharide chains that end up on the
  outside surfaces of cells (glycocalyx, basal laminae) or
  in secreted products (mucus).

  The histochemical identification of sialic acids can be
  crude - staining of the carboxylic acid, with various
  controls to exclude other reasons for basophilia. Some, but
  not all sialic acids are PAS-positive, and it is possible to
  do a special periodate oxidation that picks these out by
  not oxidizing hexoses.

  Methods for staining individual types of sialic acid are more
  subtle, exploiting the various combinations of free and
  and acetylated hydroxyl groups on C7, C8 & C9. A logically
  arranged set of combinations of simple blocking and unmasking
  tests, associated with PAS staining was developed in the
  1970s by the late Phil Reid, Chas Culling and their talented
  pupils in Vancouver. Their methods allow the recognition of
  several types of sialic acids, and were put to good use in
  studies of normal and malignant mucus-secreting cells in the
  colon. Reid & Culling also developed staining methods that
  combined their PAS variants with alcian blue at either low
  or very low pH, so that different shades of magenta, purple
  and blue showed the co-localization of various sialic acids,
  neutral mucosubstances, and weakly acidic (eg hyaluronic acid)
  or strongly acidic (eg sulphated mucus, chondroitin sulphates
  etc) ones. These elegant methods represent a pinnacle of
  achievement in the field of classical histochemistry.

  There are also various affinity-based techniques, using
  either antibodies or lectins.

  This message is quite long, but all it really says is
  that your boss must do some reading. Staining for all
  sialic acids is easy but pretty uninformative. The real
  histochemistry is also easy, but the methods and their
  controls must be carefully thought out in the light of
  the information being sought and the strengths and
  limitations of the available techniques.

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

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