FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

0.3% Acidified Potassium Permanganate (Stock)

Potassium permanganate -------------------------------------------------------                0.3 gm

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

Sulfuric acid, concentrated -----------------------------------------------------                0.2 ml

       For use dilute with equal parts of distilled water.

1% Oxalic Acid Solution

Oxalic acid ---------------------------------------------------------------------                1.0 gm

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

1% Orcein Solution

Orcein, C.I. (Ed. 1) 1242 -------------------------------------------------------                1.0 gm

Alcohol, 70% -------------------------------------------------------------------                100.0 ml

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

       Let solution stand at room temperature for at least one week before use.

• Use control slide.

1. Deparaffinize and hydrate to distilled water.
2. Place in diluted acidified potassium permanganate for 5 minutes.
3. Rinse in two changes of distilled water.
4. Place in 1% oxalic acid for 2 minutes.
5. Wash in running tap water for 1 minutes.
6. Rinse in two changes of distilled water.
7. Dip several times in 70% alcohol.
8. Place in 45 ml of orcein solution in a glass Coplin jar and microwave at power level for 1 ½ minutes.  Transfer to a 37o oven for 2 hours.
9. Rinse slides in four changes of distilled water.
10. Dehydrate in graded alcohols.
11. Clear in xylene, three or four changes.
12. Mount with synthetic resin.

Hepatitis B surface antigen (HbsAg) -------------------------------                brown (diffuse cytoplasmic)

Copper binding protein -------------------------------------------                brown, fine cytoplasmic granules

Elastic fibers --------------------------------------------------------        brown

Some orcein dyes do not give satisfactory staining results.  Dyes which are not satisfactory will either overstain the section or not adequately stain the HbsAg.  Dyes certified as orcein by the Biological Stain Commission are tested for their ability to stain HbsAg.  The stain may be done at room temperature by staining for 4-8 hours in the orcein solution.

Deodhar, K.P., et al.:  Orcein staining of hepatitis B antigen in paraffin sections of liver biopsies.  J. Clin. Path. 28:66-70, 1975.

Luna, L.G.:  A modified aldehyde fuchsin for HBAg.  Histo-Logic 8:119-120, 1978.

Shikata, T., et al.:  Staining methods of Australia antigen in paraffin sections.  Jap. J. Exp. Med. 44:25-36, 1974.