FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 µm.

SOLUTIONS:

0.05% Nile Blue Solution

Nile blue, C.I. 51180 -----------------------------------------------------------                0.05 gm

Distilled water -------------------------------------------------------------------        99.00 ml

Sulfuric acid ---------------------------------------------------------------------        1.00 ml

       Dissolve the Nile blue in the distilled water and then add the

       sulfuric acid.  The solution is stable for several months.

1.        Deparaffinize and hydrate paraffin sections to distilled water.

2.        Place slides in the Nile blue solution for 20 minutes.

3.        Rinse in four changes of distilled water.

4.        Mount with glycerin jelly.

Melanin ------------------------------------------------------------                dark green

Lipofuscin ----------------------------------------------------------                dark blue or blue-green

Nuclei and background ---------------------------------------------        blue

According to Lillie, if the sections are treated with acetone or 1% sulfuric acid after they are stained, lipofuscin will be removed but melanin will remain stained.  Therefore, when doing the stain use control slides for both melanin and lipofuscin.

Lillie, R.D.:  A Nile blue staining technic for the differentiation of melanin and lipofuscins.  Stain Technology 31:151-153, 1956.