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.

1.0% Oxalic acid

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

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

Aldehyde Fuchsin

Pararosanilin, C.I. 42500* -----------------------------------------------------                0.5 gm

Alcohol, 70% ------------------------------------------------------------------                96.0 ml

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

Paraldehyde or Acetaldehyde -------------------------------------------------                3.0 ml

       Let stand for four or five days or until stain is deep purple.         

Keeps for six to eight weeks if stored at room temperature.

          *Rosanilin, C.I. 42510, and new fuchsin, C.I. 42520, which are

closely related to pararosanilin should not be used to prepare

aldehyde fuchsin.

• 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 1.0% oxalic acid for 2 minutes.
5. Wash in running tap water for 3 minutes.
6. Rinse in three changes of distilled water.
7. Place in 70% alcohol for 30 seconds.
8. Aldehyde fuchsin for 5 to 60 minutes.  The time will depend on the age of the aldehyde fuchsin.  Longer staining times may be necessary as the solution slowly deteriorates.
9. Rinse 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 ---------------------------------------                purple (diffuse cytoplasmic)

Lipofuscin ---------------------------------------------------------        purple, cytoplasmic granules

Copper binding protein -------------------------------------------                purple, cytoplasmic granules

Elastic fibers ------------------------------------------------------                purple

Since hepatocyte and Kupfer cell intracytoplasmic granules (i.e., lipofuscin, copper binding protein) are stained with this procedure, interpretation of the presence of HbsAg has to be based on the finding of diffuse cytoplasmic staining in involved liver cells, which are usually scattered randomly throughout the parenchyma.  Lipofuscin granules tend to be perinuclear and present in all liver cells.  Copper binding protein granules are usually more coarse than lipofuscin ones, and in cells at the periphery of liver lobules or nodules.

The basic fuchsins rosanilin, C.I. 42510, and new fuchsin, C.I. 42520, are closely related to pararosanilin, C.I. 42500.  Only pararosanilin will give satisfactory staining results in this procedure.

The staining time in aldehyde fuchsin will vary considerably depending upon its age.  Fresh solution will give adequate staining in 5-10 minutes.  Solutions which are over a month old will require 60 or more minutes.  We have found that the solution keeps better if stored at room temperature than in a refrigerator. 

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

Luna, L.G.:  Recommended procedure for demonstrating hepatic B antigen (HBAg) in paraffin sections.  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.