Re: Sialidase Digestion
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
On Tue, 24 Oct 2000, Mitchell, Jeannette M. wrote:
> I have been asked to do an Alcian Blue with and without Sialidase Digestion.
> Does anyone do it or is there another procedure that is being done???
There are several histochemical methods for sialic acids. The one
you have been asked to do is probably the simplest.
1a. Immerse or cover sections in 0.1M acetate buffer, pH 5.5,
with 1 mg/ml of calcium chloride added. These will be +ve
for all acidic carbohydrates, including ones that contain
sialic acids. Leave overnight at 37C. Rinse in water.
Proceed to Step 2.
1b. Cover sections with the above solution to which has been
added 0.025 international units/ml of neuraminidase. Leave
overnight at 37C (in humid container to stop evaporation).
Rinse in water. Proceed to Step 2. In these slides sialic
acid-containing carbohydrates should be unstained, but other
acidic mucosubstances stain normally.
2. Stain with alcian blue, pH 2.5 (1% AB in 3% acetic acid)
for half an hour in the usual way. Counterstain nuclei with
something red if desired. Dehydrate, clear and mount.
There are various types of neuraminidase (= sialidase). The
enzyme from Vibrio cholerae is probably the best. Some sialic
acid groups resist sialidase, so this is not a 100% certain
diagnostic test. All sialic acid units are removed by immersing
sections in 0.3% conc sulphuric acid in water for 1 hour at 80C.
This hydrolysis is a pretty good control. You may also lose some
non-sialylated material but if you do, it doesn't show in
alcian blue-stained sections.
There are other histochemical methods that can provide more
information about sialic acids, including various variants of
the PAS method and staining with certain labelled lectins.
I can provide references for the above statements if you ask,
but alcian blue/sialidase (or acid hydrolysis) is in every
histochemistry book published since the late 1950s.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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