Solutions and Reagents:

0.5% Aqueous Periodic Acid

Methenamine Silver Stock Solution:

      3% Methenamine ---------------- 100 ml

      5% Silver nitrate ----------------- 5 ml

      Add the silver nitrate solution in small amounts to the methenamine solution, mixing after each addition. A white precipitate will form and re-dissolve on shaking. Stock solution should be clear for use. Filter the stock solution into a brown bottle. Solution is stable for several months if stored at 4 C.

Methenamine Silver Working Solution:

      Methenamine silver stock solution -------- 50 ml

      5% sodium borate (borax) solution -------- 5 ml

      Mix well. Filter and pre-heat for 10-20 minutes (Not longer, since solution will turn black after 1 hour and increase background staining) at 60 ºC prior to actual use. Allow another Coplin jar containing distilled water to preheat also.

0.2% Gold Chloride

3% Sodium Thiosulfate

Nuclear Fast Red

Procedure:

1.   Deparaffinize slides to distilled water.

2.   Oxidize in 0.5% periodic acid solution for 15 minutes at room temperature.

3.   Rinse 3 times in distilled water.

4.   Incubate slides in methenamine silver working solution for 30 min to 1 hour at 60 ºC.

5.   Rinse in hot distilled water and check microscopically.

6.   Rinse in distilled water at room temperature.

7.   Tone in gold chloride solution for 1 minute.

8.   Rinse in distilled water.

9.   Treat with sodium thiosulfate solution for 2 minutes.

10. Wash in running tap water for 10 minutes.

11. Counterstain in nuclear fast red or light green for 5 min.

12. Dehydrate, clear in xylene and coverslip using a synthetic mounting medium.

Results:

      Basement membranes, reticular fibers ------- black

      Nuclear or backgroud --------------------------- pink or green

Positive Controls:

      Skin, Kidney.