FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin or plastic sections cut at 2-3 μm.

SOLUTIONS:

0.5% Periodic Acid Solution

Periodic acid ---------------------------------------------------------------------        0.5 gm

Distilled water -------------------------------------------------------------------        100.0 ml

0.5% Thiosemicarbazide

Thiosemicarbazide --------------------------------------------------------------                0.5 gm

Distilled water -------------------------------------------------------------------        100.0 ml

5% Silver Nitrate Solution

Silver nitrate ---------------------------------------------------------------------        1.0 gm

Distilled water -------------------------------------------------------------------        20.0 ml

3% Methenamine Solution

Hexamethylenetetramine (methenamine) ------------------------------------        -----        12.0 gm

Distilled water -------------------------------------------------------------------        375.0 ml

Ethylene glycol ------------------------------------------------------------------                25.0 ml

Methenamine-Silver Nitrate Solution (Stock)

Silver nitrate, 5% solution -----------------------------------------------------                20.0 ml

Methenamine, 3% solution ----------------------------------------------------                400.0 ml

       This solution is good for about one month when stored in a

       Refrigerator at 3o-6o C.

3% Sodium Borate (Borax) Solution

Sodium borate, Na2 B4 O710 H2O ----------------------------------------------        --        3.0 gm

Distilled water --------------------------------------------------------------------        100.0 ml

Methenamine-Silver Nitrate Solution (Working)

Methenamine-silver nitrate solution (stock) ----------------------------------        -----        20.0 ml

Distilled water -------------------------------------------------------------------        20.0 ml

Sodium borate, 3% solution -----------------------------------------------------                2.0 ml

       Prepare fresh.

1% Gold Chloride (Stock)

Gold chloride ---------------------------------------------------------------------        1.0 gm

Distilled water --------------------------------------------------------------------        100.0 ml

0.2% Gold Chloride (Working)

Gold chloride, 1% ----------------------------------------------------------------        20.0 ml

Distilled water --------------------------------------------------------------------        80.0 ml

2% Sodium Thiosulfate

Sodium thiosulfate ---------------------------------------------------------------        2.0 gm

Distilled water -------------------------------------------------------------------        100.0 ml

0.1% Nuclear Fast Red Solution

(See Gomori’s Iron Stain)

1. Deparaffinize and hydrate to distilled water.  For plastic sections begin at Step 2.
2. Place in 0.5% periodic acid solution for 10 minutes for paraffin sections or for 15 minutes for plastic sections.
3. Rinse in four changes of distilled water.
4. Place in 0.5% thiosemicarbazide for 5 minutes.
5. Rinse with four changes of distilled water.
6. Place in working methenamine-silver nitrate solution and place in a 43o C water bath for 2 minutes.  Transfer to a 58o C water bath for 15 minutes.  Rinse in distilled water and check with a microscope.  The basement membranes should be dark brown.  If they are too light return to the hot methenamine-silver nitrate solution for 1 or 2 minutes.  Check slides again.  Because the staining reaction takes place rapidly care must be taken not to overstain the basement membranes.
7. Rinse with four changes of distilled water.
8. Tone in 0.2% gold chloride for 1 minutes. 
9. Rinse in two changes of distilled water.
10. Place in 2% sodium thiosulfate for 1 minutes. 
11. Rinse with four changes of distilled water.
12. Nuclear fast red solution for 3 minutes.
13. Rinse with three changes of distilled water.
14. Dehydrate paraffin processed sections in graded alcohols.  Plastic sections must be air dried then dipped in xylene and mount.
15. Clear in xylene three or four changes and mount with synthetic resin.

Basement membranes ----------------------------------------------        black

Reticulum fibers ----------------------------------------------------        black

Nuclei --------------------------------------------------------------        red

Treating the slides with thiosemicarbazide accelerates the reaction of methenamine silver with the glomerular basement membranes.  This greatly reduces the time required to achieve satisfactory staining results in comparison with Jones original method.  The addition of ethylene glycol to methenamine silver nitrate helps to stabilize the solution when it is heated.  This usually prevents the formation of a mirror of silver on the slides and Coplin jar.

This stain is used mainly in kidney biopsies, which are sometimes embedded in plastic, to demonstrate glomerular and tubular basement membranes.

Plastic sections have a tendency to come off the slides with staining procedures which require heat.  This problem can usually be avoided if the slides are laid flat on the bottom metal plate of a 60o C drying oven and are kept there for several hours.  The use of alcohols for dehydrating plastic sections must be avoided as this causes the sections to either partially or completely come off the slide.

Gomori, G.:  A new histochemical test for glycogen and mucin.  Amer. J. Clin. Path. 10:177-179, 1946.

Jones, D.B.:  Inflammation and repair of glomerulus.  Amer. J. Path. 27:991-1009, 1951.

Hayashi, J.:  Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.  Stain Technol. 64:185-190, 1989.