FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 µm.

SOLUTIONS:

Phosphate Buffer. pH 6.0

Sodium phosphate, dibasic -----------------------------------------------------                0.128 gm
Potassium phosphate, monobasic ----------------------------------------------                0.872 gm
Distilled water ------------------------------------------------------------------                100.0 ml

Hyaluronidase Digestion Solution

Hyaluronidase, testicular (Sigma) -----------------------------------------------                0.02 gm
Phosphate buffer, pH 6.0 -------------------------------------------------------                40.00 ml

Mix just before use.

3% Acetic Acid Solution

(see Microwave Alcian Blue Method)

1.0% Alcian Blue Solution

(see Microwave Alcian Blue Method)

0. 1% Nuclear Fast Red Solution

(see Microwave Alcian Blue Method)

• Use control slide (duplicate section).

1.        Deparaffinize and hydrate duplicate sections to distilled water.

2.        Place the control and requested section in 40 ml of hyaluronidase digestion solution in a glass Coplin jar and microwave at power level 1 (60W) for 1 minute.  Transfer to a 37° C oven for 1 hour.  Place the duplicate control and requested slides in 40 ml of phosphate buffer, pH 6.0 in a glass Coplin jar and microwave at power level 1 for 1 minute.  Transfer to a 37o C oven for 1 hour.

3.        Rinse all the slides in four changes of distilled water.
4.        Place in 3% acetic acid solution for 3 minutes.

5.        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.

6.        Rinse in three changes of distilled water.
7.        Nuclear fast red solution for 3 minutes.
8.        Rinse in three changes of distilled water.
9.        Dehydrate in graded alcohols.
10.        Clear in three or four changes of xylene.
11.        Mount with synthetic resin.

Staining attributable to hyaluronic acid, chrondrotin 4 sulfate, or chrondrotin 6 sulfate is selectively eliminated.

The alcian blue method with and without hyaluronidase digestion has some value in differentiating mesotheliomas from carcinomas. Some mesotheliomas contain hyaluronic acid and therefore can be stained with alcian blue. If the alcian blue positivity is destroyed following prior incubation with hyaluronidase this helps in establishing the diagnosis of mesothelioma.

When heating the alcian blue solution with microwave irradiation the top portion of the solution is warmer by 10-15° 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.

Luna, L.G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., New York, McGraw-Hill, 1968, pp. 171-172.