FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

Citric Acid-Sodium Citrate Solution (Stock)

Citric acid, monohydrate -------------------------------------------------------                0.2 gm

Sodium citrate, dihydrate ------------------------------------------------------                0.2 gm

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

       The pH of this solution is approximately 3.8.  Store in a refrigerator

       at 3-6o C.

Citric Acid-Sodium Citrate Solution (Working)

Citric acid-sodium citrate (stock) ---------------------------------------------                1.5 ml

Distilled water ----------------------------------------------------------------                150.0 ml

       The pH of this solution is approximately 3.8.

0.5% Silver Nitrate Solution

Silver nitrate ---------------------------------------------------------------------        0.2 gm

Citric acid-sodium citrate, working solution --------------------------------        -----                40.0 ml

       Prepare fresh.

Bodian’s Developer

Sodium sulfite (anhydrous) -------------------------------------------------------        2.0 mg

Hydroquinone -------------------------------------------------------------------        0.4 gm

Distilled water -------------------------------------------------------------------        40.0 ml

0.1% 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, add a few grains of thymol as

       a preservative.

• Use control slide.

1. Deparaffinize and hydrate to acidulated water pH 3.8.
2. Place sections in 0.5% silver nitrate solution and place in a 43o C water bath for 2 minutes.  Transfer to a 58o C water bath for 2 hours.  The solution must be freshly prepared.
3. Rinse in five changes of distilled water.
4. Transfer sections to Bodian’s developer, which has been previously heated in a water bath to 58o C for 2 minutes.
5. Rinse in five changes of distilled water.
6. Reimpregnate in the same 0.5% silver nitrate solution in the 58o C water bath for 3 minutes.
7. Rinse in five changes of distilled water.
8. Place again in Bodian’s developer in the 58o C water bath for 2 minutes.
9. Rinse in five changes of distilled water.
10. Place in the same 0.5% silver nitrate solution in the 58o C water bath for 2 minutes.
11. Rinse in five changes of distilled water.
12. Transfer slides to the Bodian’s developer in the 58o C water bath for 2 minutes.
13. Rinse in four changes of distilled water.
14. Counterstain with 0.1% nuclear fast red for 1 minute.
15. Rinse in two changes of distilled water.
16. Dehydrate in graded alcohols.
17. Clear in three or four changes of xylene and mount with synthetic resin.

Neuroendocrine cell granules (argentaffin and argyrophil) in various organs stain dark brown to black.  Nuclei are red and the background a light yellow-orange.

This staining method has been found, by Smith and Haggitt, to be a reliable and sensitive silver method for demonstrating secretory granules in carcinoid tumors.  They, and others, have found the method to produce superior staining results when compared to the accepted method of Gremelius.

Churukian, C.J. and Schenk, E.A.:  A modification of Pascual’s argyrophil method.  J. Histotechnology 2:102-103, 1979.

Pascual, J.S.:  A new method for easy demonstration of argyrophil cells.  Stain Technol. 51:231-235, 1976.

Smith, D.M. and Haggitt, R.C.:  A comparative study of generic stains for carcinoid secretory granules.  Amer. J. Surg. Pathol. 7:61-68, 1983.