Methenamin Silver Method for Argentaffin Cells

EMS26481

Components:

Weigert's Iodine Solution

Sodium Thiosulfate 5% Aqueous

Silver Nitrate 5% Aqueous

Methenamine 3% Aqueous

Borate Buffer pH 7.8

Gold Chloride 0.1% Aqueous

Safranin O Solution 0.1%

Fixation:

Formalin, Bouin’s or other Formalin containing fixatives. Dichromates and mercury salts impair contrast between granules and background.

Sections:

Cut paraffin at 3-5µm sections.

Staining Procedures:

1. Deparaffinise and hydrate slides to distilled water.
2. Treat for 10 minutes in Weigert’s Iodine followed by bleaching for 10 minutes in Sodium Thiosulfate, 5%. Wash for 10 minutes in running water. Rinse in two changes in distilled water.
3. Place section in chemically clean coplin jars containing the buffered methenamine-silver nitrate solution at room temperature and place in 60°C paraffin oven for 3 – 3.5 hours. (preheating the solution to 60°C reduces impregnation time by ½ to 1 hour). Rinse in distilled water.
   1. * prepare methenamine-silver nitrate by mixing:
      Silver Nitrate, 5% - 5ml
      Methenamine, 3% - 100ml
      For working buffered stain add 8 ml of Borate Buffer, pH 7.8 to 30ml of methenamine-silver nitrate. Use chemically clean glassware throughout.
4. Tone for 10 minutes in Gold Chloride, 0.1%. Rinse in distilled water.
5. Fix in Sodium Thiosulfate, 5% for 2 minutes. Wash in running water for 5 minutes
6. Counterstain in Acetic Safranin O, 0.1% for 5 minutes.
7. Dehydrate with Acetone, clear in Xylene and mount.

Stain Results:  

References:

Clark, G, ed.: Staining Procedures, 3rd ed.: Baltimore: Williams & Wilkins Co., 1973, p. 161

Gomori, G.: Arch Path., 45:48, 1948

Burtner, H.J. and Lillie, R.D., Stain Tech., 24:225-7, 1949.