FIXATION:        10% buffered neutral formalin for 48 hours or longer.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

Modified Weigert’s Iron Hematoxylin

(See Gomori’s One Step Trichrome Method)

0.5% Acid Alcohol Solution

(See Truant’s Method for Acid Fast Bacilli)

Picro-Orange G Solution

Picric acid, saturated aqueous -------------------------------------------------                100.0 ml

Orange G, C.I. 16230 -----------------------------------------------------------        1.0 gm

Ponceau-Fuchsin Solution

Acid fuchsin, C.I. 42685 -------------------------------------------------------                0.5 gm

Ponceau 2R (Ponceau Xylidine) C.I. 16150 ------------------------------------                0.5 gm

Distilled water -------------------------------------------------------------------        99.0 ml

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

*Phosphomolybdic-Phosphotungstic Acid Solution

Phosphomolybdic acid ----------------------------------------------------------                2.5 gm

Phosphotungstic acid -----------------------------------------------------------                2.5 gm

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

       *This solution is the same one used in Masson’s trichrome stain.

*Aniline Blue Solution

Aniline blue, C.I. 42755 --------------------------------------------------------        -        1.0 gm

Distilled water -------------------------------------------------------------------        40.0 ml

Acetic acid -----------------------------------------------------------------------        0.8 ml

       *This solution is the same one used in Masson’s trichrome stain.

• Use control slide.

1. Deparaffinize and hydrate to distilled water.
2. Modified Weigert’s iron hematoxylin for 5 minutes.
3. Wash in running water and rinse in distilled water.
4. Place in acid alcohol for 5 seconds.
5. Wash in running water for 1 minute and rinse in distilled water.
6. Picro-orange G solution for 3 minutes.
7. Rinse in three changes of distilled water.
8. Ponceau-fuchsin for 3 minutes.
9. Rinse in three changes of distilled water.
10. Differentiate in phosphomolybdic acid-phosphotungistic acid solution for 3 minutes.
11. Rinse in three changes of distilled water.
12. Aniline blue solution for 2 minutes.
13. Rinse in three changes of distilled water.
14. Dehydrate in graded alcohols and clear in xylene, three or four changes.
15. Mount with synthetic resin.

Fibrin ----------------------------------------------------------------        clear bright red

Muscle --------------------------------------------------------------        red

Platelets -------------------------------------------------------------                leaden blue to navy blue

Collagen ------------------------------------------------------------        blue

Erythrocytes --------------------------------------------------------        yellow or orange

This is a difficult stain to evaluate.  Results are occasionally erratic and platelet staining tends to be variable.

Carstairs, K.C.:  The identification of platelets and platelet antigens in histological sections.  J. Path. Bact. 90:225, 1965.