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.

  4. * 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.

  5. Tone for 10 minutes in Gold Chloride, 0.1%. Rinse in distilled water.

  6. Fix in Sodium Thiosulfate, 5% for 2 minutes. Wash in running water for 5 minutes

  7. Counterstain in Acetic Safranin O, 0.1% for 5 minutes.

  8. Dehydrate with Acetone, clear in Xylene and mount.

Stain Results:  

Argentaffin Cells

Black

Coarse connective tissue of the submucosa

Variable amount of blackening by 3-3.5 hour of impregnation

Granules of eosinophil leukocytes, nuclei, smooth muscle & surface epithelium

Show additional blackening after incubation beyond 3-3.5 hour.

Granules of mast cells

Remain red after nuclei & reticulum are blackened

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.