FIXATION:        10% buffered neutral formalin or Bouin’s.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

Bouin’s Solution

Picric acid, saturated aqueous -------------------------------------------------                75.0 ml

Formalin, concentrated, 37-40% ----------------------------------------------                25.0 ml

Acetic acid, glacial --------------------------------------------------------------                5.0 ml

Modified Weigert’s Iron Hematoxylin

Solution A

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

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

Solution B

Ferric chloride, FeCl3, 6H20, 62% aqueous -----------------------------------                4.0 ml

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

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

Working Modified Weigert’s Iron Hematoxylin

Equal parts of Solution A and Solution B

Biebrich Scarlet-Acid Fuchsin Solution

Biebrich scarlet, C.I. 26905 ----------------------------------------------------                0.45 gm

Acid fuchsin, C.I. 42685 -------------------------------------------------------                0.05 gm

Acetic acid, glacial --------------------------------------------------------------                0.50 gm

Distilled water ------------------------------------------------------------------                50.0 ml

Phosphomolybdic-Phosphotungstic Acid Solution

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

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

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

Aniline Blue Solution

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

Acetic acid, glacial --------------------------------------------------------------                0.8 ml

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

1% Acetic Acid Solution

Acetic acid, glacial --------------------------------------------------------------                1.0 ml

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

1. Deparaffinize and hydrate to distilled water.
2. Mordant in Bouin’s solution which has been preheated to 58o C for 15 minutes.
3. Wash in running water until the yellow color disappears and rinse in two changes of distilled water.
4. Modified Weigert’s iron hematoxylin for 5 minutes.
5. Wash briefly in running water and rinse in two changes of distilled water.
6. Place in 0.5% hydrochloric acid in 70% alcohol for 5 seconds.
7. Wash in running tap water for 30 seconds and rinse in two changes of distilled water.
8. Biebrich scarlet-acid fuchsin solution for 5 minutes.
9. Rinse in three changes of distilled water.
10. Phosphomolybdic-phosphotungstic acid solution for 5 minutes.
11. Aniline blue solution for 20 minutes.
12. Rinse in distilled water.
13. Acetic acid solution for 10 seconds.
14. Rinse in two changes of distilled water.
15. Dehydrate in graded alcohols.
16. Clear in three or four changes of xylene.
17. Mount with synthetic resin.

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

Cytoplasm, keratin, muscle fibers and fibrin -------------------------        red

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

Treatment of the tissue sections with hot Bouin’s solution is essential in order to obtain satisfactory results.  Some authors recommend treating the tissue section for 30-60 minutes in hot Bouin’s.  This sometimes causes tissue sections to become detached from the slides.  The 15-minute exposure to hot Bouin’s is adequate with much less tendency for tissue sections to detach from the slides.

This stain is mainly ordered for kidney biopsies.  The Gomori-trichrome method is used for other tissues because it is easier to perform and provides equally satisfactory staining of collagen and muscle.

Masson, P.J.:  Some histological methods.  Trichrome stainings and their preliminary technique.  J. Techn. Methods 12:75-90, 1929.