FIXATION:        10% buffered neutral formalin.  Bouin’s fixative should not be used.

TECHNIQUE:        Paraffin sections cut at 5 μm or undecalcified, plastic embedded, bone marrow biopsies cut at 4 μm.

SOLUTIONS:

Acidified Potassium Permanganate

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

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

Sulfuric acid ---------------------------------------------------------------------        0.2 ml

       This solution is good for about two days.

1% Potassium Metabisulfite

Potassium metabisulfite --------------------------------------------------------                1.0 gm

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

10% Silver Nitrate

Silver nitrate ---------------------------------------------------------------------        1.0 gm

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

       Store in a refrigerator at 3-6o C.

10% Potassium Hydroxide

Potassium hydroxide -----------------------------------------------------------                10.0 gm

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

Ammoniacal Silver Solution

       To 10 ml of 10% silver nitrate add 2.5 ml of 10% potassium hydroxide.  Then

       add concentrated ammonium hydroxide, drop by drop with constant shaking,

       until the precipitate just dissolves.  Carefully add 10% silver nitrate, drop by

       drop, while shaking the container continuously, until the last drop causes the

       solution to become slightly cloudy.  Make up the solution to 50 ml with

       distilled water.  This solution is stable for about 7 days if stored in a

       refrigerator at 3-6o C.

10% Formalin

Formaldehyde, 38-40% --------------------------------------------------------                10.0 ml

Distilled water -----------------------------------------------------------------                90.0 ml

0.2% Gold Chloride

Gold chloride --------------------------------------------------------------------        0.2 gm

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

0.2% Sodium Thiosulfate

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

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

0.1% Nuclear Fast Red

(see Pearl’s method for iron)

1. Deparaffinize and hydrate to distilled water.
2. Oxidize with acidified potassium permanganate for 3 minutes.
3. Rinse with three changes of distilled water.
4. Reduce with 1% potassium metabisulfite for 1 minute.
5. Wash with running tap water for three minutes and rinse with four changes of distilled water.
6. Ammoniacal silver solution for two minutes.
7. Rinse with three changes of distilled water.
8. Reduce in 10% formalin for 1 minute.
9. Wash with running tap water for 1 minute and rinse with two changes of distilled water.
10. Tone in 0.2% gold chloride for 30 seconds.
11. Rinse with two changes of distilled water.
12. Fix in 2% sodium thiosulfate for 1 minute.
13. Wash well with tap water and rinse with two changes of distilled water.
14. Counterstain with 0.1% nuclear fast red for 3 minutes.
15. Rinse with three changes of distilled water.
16. Dehydrate in graded alcohols.
17. Clear in xylene, three or four changes.
18. Mount with synthetic resin.

Reticulum -----------------------------------------------------------                black

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

When preparing the ammoniacal silver solution it is important to use the smallest amount of ammonium hydroxide necessary to dissolve the precipitate resulting from the addition of potassium hydroxide to the silver nitrate.  If too much ammonium hydroxide is used, not only will there be unsatisfactory staining of reticulum but sections are likely to become detached from the slides. 

We have observed that silver nitrate crystals stored at room temperature will gradually take on a grayish-violet color.  This does not occur when silver nitrate is kept refrigerated, even after several years.  Solution made with silver nitrate which has become discolored are usable but from our observations, seem to be less stable than those prepared with non-discolored silver nitrate.

After preparing the ammoniacal silver solution, we place it in the refrigerator.  Just prior to use the solution is brought to room temperature and immediately after use it is returned to the refrigerator.  On a number of occasions we have observed that reticulum fibers stain better after the ammoniacal silver has aged in the refrigerator for a few days.  After a few days the solution begins to take on a pale yellow-brown color which may or may not become progressively darker.  This colorization does not appear to adversely affect the quality of the staining results.  By using the same ammoniacal silver solution for several days we have been able to greatly reduce our consumption of silver nitrate with considerable cost savings.  Extreme care should be exercised in the preparation and use of ammoniacal silver.  Store ammoniacal silver in a refrigerator to avoid the formation of explosive compounds.  If ammoniacal is exposed to direct sunlight it will explode.

Churukian, C.J.:  Modified Gomori’s method for staining reticulum and collagen.  Histo-Logic 2:23, 1972.

Churukian, C.J.:  Prolonging the shelf-life of solutions containing silver nitrate.  Histo-Logic 10:147, 1980.

Gomori, G.:  Silver impregnation of reticulum in paraffin sections.  Amer. J. Clin. Path. 13:993-1002, 1937.