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Methenamine Silver (Gomori PAMS) Staining Protocol
for Reticular Fibers and Basement Membranes
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