Re: sialic acid procedure
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> (by way of Marvin Hanna) |
To: | histonet@histosearch.com |
Reply-To: | |
Content-Type: | text/plain; charset="us-ascii" |
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out of date) produced only by animals and are incorporated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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