FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

25% Trichloroacetic Acid

Trichloroacetic acid -------------------------------------------------------------                25.0 gm

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

10% Ferric Chloride Solution

Ferric chloride -------------------------------------------------------------------        10.0 gm

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

Fouchet’s Reagent

25% trichloroacetic acid -------------------------------------------------------                36.0 ml

10% ferric chloride -------------------------------------------------------------                4.0 ml

Van Gieson’s Stain

1% acid fuchsin, aqueous ------------------------------------------------------                10.0 ml

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

1. Deparaffinize and hydrate to distilled water.
2. Stain in Fouchet’s reagent for 10 minutes.
3. Wash in running water, then in distilled water.
4. Stain in Van Gieson’s stain for 2 minutes.
5. Dehydrate in 95% alcohol, three changes in Coplin jars.
6. Complete dehydration in absolute alcohol, three changes.
7. Clear in xylene, three or four changes.
8. Mount with synthetic resin.

Biliverdin -----------------------------------------------------------        green

Collagen ------------------------------------------------------------        red

Muscle --------------------------------------------------------------                yellow

Bilirubin is oxidized to biliverdin and stains olive drab green to emerald green, depending upon the concentration of bilirubin.

Hall, M.J.:  A staining reaction for bilirubin in sections of tissue.  Amer. J. Clin. Path. 34:313-316, 1960.