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FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

3% Acetic Acid Solution

Acetic acid, glacial --------------------------------------------------------------        -        3.0 ml
Distilled water --------------------------------------------------------------------        97.0 ml

1.0% Alcian Blue Solution

Alcian blue 8GX, C.I. 74240 ----------------------------------------------------                1.0 ml

3% acetic acid -------------------------------------------------------------------               100.0 ml

       Filter and add a few crystals of thymol.

Nuclear Fast Red Solution

Dissolve 0.1 gm nuclear fast red in 100 ml of 5% solution of aluminum

sulfate with aid of heat.  Cool, filter and add a few grains of thymol as

a preservative.

1.        Deparaffinize and hydrate to distilled water.
2.        Place in 3% acetic acid for 3 minutes.

3.        Place in 40 ml of 1.0% alcian blue solution in a glass Coplin jar and microwave at power level 1 (60W) for 3 minutes.  Dip the slides up and down several times and allow them to remain in the hot solution (65° C) for 5 minutes.

4.        Wash in running tap water for 1 minute and rinse in distilled water.
5.        Nuclear fast red solution for 3 minutes.
6.        Rinse in three changes of distilled water.
7.        Dehydrate in graded alcohols.
8.        Clear in three or four changes of xylene.
9.        Mount with synthetic resin.

Acidic sulfated mucosubstances -------------------------------------        blue

Cryptococcus -------------------------------------------------------        blue

Nuclei----------------------------------------------------------------        red

No single staining solution of alcian blue colors all polyanions maximally, but the above procedure, done at pH 2.5, stains all except the most strongly sulfated mucopolysaccharides, and is very useful for demonstrating goblet cell mucins in the respiratory tract and in small and large bowel, as well as various components of skin, heart valve.s, large arteries and cartilage.

Alcian blue probably find sits greatest usefulness in combination stains such as Alcian blue, PAS, and the Kreyberg method.

For a more detailed discussion of various uses of alcian blue see: Schenk, E.A. and Mowry, R.W.: Alcian Blue. J. Histotech., June 1983, pp. 65-69.

When heating the alcian blue solution with microwave irradiation the top portion of the solution is warmer by 10-15° C than that near the bottom of the Coplin jar. Therefore, in order to equalize the temperature of the solution the slides are dipped up and down. This assures uniformity of staining throughout the tissue sections.

The stain may be performed at room temperature by staining in the alcian blue solution for 30 minutes.

McManus, J.F. and Mowry, R.W.: Staining Methods Histologic and Histochemical, New York, Paul B. Hoeber, 1960, pp. 137-138.