FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

Weigert’s Iron Hematoxylin (Concentrated)

Solution A

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

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

Solution B

Ferric chloride, Fe Cl3 6 H20 62% --------------------------------------------                4.0 ml

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

Hydrochloric acid, concentrated ----------------------------------------------                1.0 ml

       For use mix equal parts of each solution.

Mucicarmine Solution (Stock)

Carmine, C.I. 75470 ------------------------------------------------------------                1.0 gm

Aluminum hydroxide -------------------------------------------------------------        1.0 gm

50% alcohol ---------------------------------------------------------------------        100.0 ml

       Mix and then add:

Aluminum chloride hexahydrate ------------------------------------------------                1.0 gm

       Heat and boil 3 minutes.  Cool and make up to original volume with

       50% alcohol.  Filter through Pyrex glass wool.  This stock solution is

       stable for several months when stored in a refrigerator at 3-6o C.

       To prepare the working solution add 30 ml. of distilled water to 10 ml.

       of the stock solution.

0.2% Tartrazine Solution

Tartrazine, C.I. 19140 ----------------------------------------------------------                0.2 gm

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

Acetic acid ----------------------------------------------------------------------        0.2 ml

1. Deparaffinize and hydrate to distilled water.
2. Weigert’s iron hematoxylin for 1 minute.
3. Wash briefly in running water.
4. Decolorize with 0.5% hydrochloric acid in 70% alcohol for 10 seconds.
5. Wash in running tap water for 1 minute and rinse in distilled water.
6. Place in diluted mucicarmine solution for 90 minutes or longer (2-3 hours) if a more intense stain is desired.
7. Rinse quickly in distilled water.
8. Place in tartrazine solution for 10 seconds.
9. Rinse quickly in distilled water.
10. Dehydrate in graded alcohols.
11. Clear in xylene, three or four changes.
12. Mount with synthetic resin.

Mucin ----------------------------------------------------------------        deep rose to red

Capsule of Cryptococcus -----------------------------------------                deep rose to red

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

Other tissue elements ---------------------------------------------                yellow or orange

This method gives better staining results than Mayer’s mucicarmine method.  It will demonstrate acidic mucopolysaccharides similar to those stained with Alcian blue (pH 2.5) and with colloidal iron.  For many years we used metanil yellow as the counterstain in this method.  This counterstain has a slight masking effect upon the stained mucin.  We found that tartrazine produces a lighter background stain which allows for better visualization of the mucin.

Culling, C.F.A.:  Handbook of Histopathological Technique, 3rd ed., Butterworths, London, 1974, pp. 304-305.

Luna, L.G.:  Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., New York, McGraw-Hill, 1968, pp. 161-162.