Aim: To demonstrate the presence of argentaffin granules.

Principle: Granules in argentaffin cells reduces ammoniacal silver solution to metallic silver. This histo-chemical reaction is due the presence of 5 hydroxy tryptamine(5HT).The 5HT must be converted to tetrahydri-carbolin derivative
by formalin fixation before reactions can be demonstrated.

Control: skin or any positive tissue

Reagents 

1. Stock 10% silver nitrate solution:-

Silver nitrate A R grade        10gm
Distilled water                       100ml

2. Fontana masson silver nitrate solution

To 50 ml of 10% silver nitrate solution, add one or two drops of ammonium hydroxide. The first drop itself will cause a brown precipitate. Continue to add ammonia solution drop by drop just until the solution is clear. From stock 10% silver nitrate solution, add a little more solution drop by drop dissolving the initial precipitate and stop when a permanent faint turbidity is attained. Let it stand overnight to settle. Before use, decant silver solution, filter and dilute with an equal amount of distilled water. Prepare the fresh solution each time.

3. Gold chloride solution:-

Gold chloride                      1gm
Distilled water                    500ml
Keep the solution in refrigerator.

4.  Sodiumthiosulfate                              5gm
     Distilled water                                     100ml

Procedure:-

1. Bring sections to distilled water
2. Treat with Fontana silver nitrate solution for 1 hour at 56-58IN0C
ammoniacal silver solution in a closed jar 15 mins
3. Check microscopically and repeat step 2 if necessary                                                                                                                  4. Wash well in distilled water
5. Tone with gold chloride 2 minutes
6. Wash well with distilled water
7. Fix in 2% aq sodium thiosulphate 2 minutes
8. Wash well with distilled water
9. Counter-stain with neutral red stain 1 minutes
10. Rinse in distilled water
11. Rapidly dehydrate well in absolute alcohol, clear and mount.

Results:-

Melanin                                                 black
Argentaffin cell granules                    black
Some lipofuscins                                black
Chromaffin                                           black
Nuclei                                                    red

Note:- 
*Formalin fixation is essential for argentaffin substances, but not critical formelanin.
*A known positive control section must be used to ensure correct demonstration
  has been achieved.
*Time of the ammoniacal silver impregnation depends upon the tissue component to be demonstrated. At room         temperature, melanin will require 12 hrs, argentaffin 24 hrs. At 60°C melanin blackens within 20 minutes, argentaffin     requires approximately 40 minutes. Excessive heat over long periods may cause the silver solution to precipitate, give   non-specific background deposits, and cause precipitation of silver on connective tissue fibres.
*Ammoniacal silver solutions can be explosive when allowed to dry. Immediately after use neutralise the silver solution with saturated sodium chloride and discard.