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FIXATION:        10% buffered neutral formalin.

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

90% ethyl alcohol --------------------------------------------------------------                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

       For use mix equal parts of Solution A and Solution B.

Trichrome Stain

Chromotrope 2R, C.I. 16570 --------------------------------------------------                0.6 gm

Aniline blue, C.I. 42755 --------------------------------------------------------                0.3 gm

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

Phosphotungstic acid -----------------------------------------------------------                0.8 gm

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

       Fast green F.C.F. or light green S.F. may be substituted for aniline

       blue if it is more desirable to have collagen stained green.

0.5% Acetic Acid

Acetic acid, glacial --------------------------------------------------------------                0.5 ml

Distilled water -------------------------------------------------------------------        99.5 ml

1.       Deparaffinize and hydrate to distilled water.

2.         Place in Bouin’s solution which has been preheated to 58o C for 10 minutes.

3. Wash in running tap water until the yellow color disappears and then rinse in distilled water.
4. Modified Weigert’s iron hematoxylin for five 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. Trichrome stain for 15 minutes.
9. Acetic acid water, 0.5% for 10 seconds and rinse in two changes of distilled water.
10. Dehydrate in graded alcohols.
11. Clear in xylene, three or four changes.
12. Mount with synthetic resin.

Muscle fibers -------------------------------------------------------                red

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

Nuclei ---------------------------------------------------------------                blue to black

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

Treatment with hot Bouin’s solution is necessary to obtain satisfactory staining of muscle and collagen.  Without this pretreatment color differentiation of muscle and collagen will be very poor.

Plastic sections may be stained with this method, but the quality of the stain tends to be less satisfactory than with paraffin sections.

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

Gomori, G.:  A rapid one-step trichrome stain.  Am. J. Clin. Path. 20:661-664, 1950.