FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

0.3% Acetic Acid Solution

(See Microwave Alcian Blue Method)

1% Alcian Blue Solution

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

Distilled water ------------------------------------------------------------------                100.0 ml

Acetic acid ---------------------------------------------------------------------                1.0 ml

Ammonia-Alcohol Solution

Alcohol, 95% ------------------------------------------------------------------                90.0 ml

Ammonium hydroxide, 28% ----------------------------------------------------                10.0 ml

Weigert’s Iron Hematoxylin, Concentrated

Solution A

Hematoxylin crystals, C.I. 75290 ---------------------------------------------                2.0 gm

Alcohol, 90% ----------------------------------------------------------------                100.0 ml

Solution B

Ferric chloride, 62% aqueous -------------------------------------------------                4.0 ml

Distilled water -----------------------------------------------------------------                95.0 ml

Hydrochloric acid, conc. -------------------------------------------------------                1.0 ml

       To make up the working solution, mix equal parts of Solution A

       and Solution B.

Acid Alcohol Solution

Alcohol, 70% ------------------------------------------------------------------                100.0 ml

Hydrochloric acid, conc. -------------------------------------------------------                0.5 ml

1% Erythrosin B Solution

Erythrosin B, C.I. 45430 -------------------------------------------------------                1.0 gm

Distilled water ------------------------------------------------------------------                100.0 ml

Alcoholic Metanil Yellow Solution

Metanil yellow, C.I. 13065 ----------------------------------------------------                0.5 gm

Alcohol, absolute ---------------------------------------------------------------                100.0 ml

Filter and add acetic acid -------------------------------------------------------                1.0 ml

1. Deparaffinize and hydrate to distilled water.
2. Place in 3% acetic acid for 3 minutes.
3. Place in 40 ml of 1% alcian blue solution in a glass Coplin jar and microwave at power level 1 (60W) for 3 minutes.  Dip the slides up and down and allow them to remain in the hot solution for 5 minutes.
4. Rinse in three changes of distilled water.
5. Place in ammonia-alcohol solution for 10 minutes.
6. Wash well with tap water and rinse in distilled water.
7. Place in Weigert’s iron hematoxylin solution for 45 seconds.
8. Wash with distilled water.
9. Acid alcohol solution for 10 seconds.
10. Rinse in four changes of distilled water.
11. Place in erythrosin B solution for 5 minutes.
12. Rinse quickly in one change of distilled water.
13. A few quick dips in two changes of 95% alcohol followed by two changes of absolute alcohol.
14. Place in alcoholic metanil yellow for 30 seconds.
15. Dehydrate with four changes of absolute alcohol.
16. Clear in three or four changes of xylene and mount with synthetic resin.

Keratin ---------------------------------------------------------------                red or orange red

Mucin ----------------------------------------------------------------        green or bluish green

Nuclei ---------------------------------------------------------------                black

Collagen ------------------------------------------------------------                orange

Muscle --------------------------------------------------------------                various shades of red and orange

Erythrocytes --------------------------------------------------------        red

This method has been useful in distinguishing between keratinizing and mucus producing tumors and in identifying tumors which contain both elements.  This method has been used most often to stain tumors of the lung, gastrointestinal tract, breast, bladder, prostate and female genital tract.  It is used to stain cytologic smears, especially bronchial aspirations.

The modification of Weigert’s iron hematoxylin used in this method results in clear, sharp nuclear detail which is not affected by treatment with the other staining solutions.  Following staining with alcian blue the method uses ammonia-alcohol which converts the alcian blue into the insoluble pigment monastral fast blue.  If the sections are not treated with ammonia-alcohol after being stained with alcian blue, there will be a significant loss of stain from the acid mucopolysaccharides.

For several years we used saffron in this staining method.  During the past few years the cost of saffron has greatly increased, therefore, a search was made for a substitute dye.  Metanil yellow was found to be satisfactory in alcoholic solution when it was acidified with acetic acid.

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

Churukian, C.J. and Schenk, E.A.:  A modification of Kreyberg’s method for demonstrating keratin and mucin.  J. Histotechnology 7:146-148, 1984.

Elbadawi, A.:  Hexachrome modification of Movat’s stain.  Stain Technol. 51:249-253, 1976.

Kreyberg, L.:  Main histological types of primary epithelial lung tumors.  Brit. J. Cancer 25:206-210, 1961.