FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Cut paraffin sections at 5 μm.

SOLUTIONS:

5% Silver Nitrate Solution

Silver nitrate ---------------------------------------------------------------------        0.5 gm

Distilled water -------------------------------------------------------------------        10.0 ml

2% Sodium Thiosulfate Solution

Sodium thiosulfate --------------------------------------------------------------                2.0 gm

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

Nuclear Fast Red (Kernechtrot) Solution

       Dissolve 0.1 gm nuclear fast red in 100 ml of 5% solution of aluminum

sulfate with the aid of heat.  Cool, filter, and add a few crystals of thymol

as a preservative.

• Use control slide.

1. Deparaffinize and hydrate to to 95% alcohol and air dry the slides.
2. Place slides on a staining rack and flood the sections with 5% silver nitrate.  Expose the slides to an ultraviolet lamp for 10 minutes.
3. Rinse with four changes of distilled water.
4. Sodium thiosulfate solution for 1 minute.
5. Rinse with four changes of distilled water.
6. Nuclear fast red solution for 3 minutes.
7. Rinse with three changes of distilled water.
8. Dehydrate through graded alcohols.
9. Clear in xylene, three or four changes.
10. Mount with synthetic resin.

Calcium salts -------------------------------------------------------        black

Nuclei --------------------------------------------------------------        red

Cytoplasm ----------------------------------------------------------                light pink

Some staining manuals and texts indicate the use of direct sunlight for 30-60 minutes in Step 2.  Direct sunlight is not always available and ultraviolet light works just as well in much less time.

Luna, L.:  Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd edition, New York, McGraw-Hill Book Company, Blakiston Division, 1968, pp. 176-177.

Mallory, F.B.:  Pathological Technique, New York, Hafner Publishing Company, 1961, p. 144.