Silver Stain for Neurodegenerative disease

1. Bielschowsky’s modified method for nerve fibre tangles

Reagents – commercial source unknown, unless specified:

3. Ammoniacal silver solution

• To the 20% aqueous silver nitrate in which the slides were sensitised, add strong ammonia dropwise, stirring vigorously until the precipitate dissolves and the solution becomes clear. Add 2 more drops of strong ammonia.

Method:

1. Bring sections to distilled water.
2. Sensitise in 20% silver nitrate for 20 minutes.
3. Place in distilled water and hold, whilst preparing ammoniacal silver solution.
4. In the dark, impregnate in ammoniacal silver solution for 15 minutes.
5. Immerse slides in ammoniacal water.
6. Add 0.5 mL developer to the jar containing the slides, stir solution.
7. Impregnate with this adjusted ammoniacal silver solution – allow the slides to remain in this solution until the fibres are black and the background is tan. This development usually takes about 3 to 5 minutes – please check microscopically.
8. Wash well with distilled water.
9. Fix with sodium thiosulphate for 5 minutes.
10. Wash well with distilled water.
11. Dehydrate, clear and mount.

Results:

Senile plagues, neurofibrillary tangles – black

Background – tan

2. Garvey silver method

Reagents – commercial source unknown, unless specified:

3. 0.5% Ammoniacal silver solution

• Place 20 mL freshly made 1% silver nitrate in a glass conical flask. Add 10% ammonium hydroxide drop by drop until initial dark precipitate becomes lightly straw coloured.
• Note: Do not let the precipitate dissolve completely. If it has you have gone too far. Add a few drops (~ 5 mL) of 1% silver nitrate until a light precipitate returns. Dilute to 40 mL with deionized water. Mix thoroughly.

• Heat the water in a microwave until warm (time depends on quantity – e.g. 30 seconds for 100 mL). Measure the hydroquinone wearing a mask and dissolve in warm water. Or use hot tap water to warm the solution in the flask. Cover in Para film. Set aside a 25 mL cylinder for measuring the hydroquinone for the reducer.

• Mix on stirrer (several hours). A gum will form at the bottom of the beaker which will not dissolve. Filter and throw out gum. Wash all glassware in 100% alcohol.

• For the reducer (following step), set aside a 50 mL cylinder washed in 100% alcohol to use for the gum mastic.

• Heat the hydroquinone till warm and combine with the gum mastic in a 50 mL Coplin jar. Just before use add 4 mL of the 1% silver nitrate into the solution. Mix well. Solution turns White.

• Note: if the gold chloride is taking a long time to turn the slides purple/grey, or there is sediment in the bottom of the Coplin jar, throw it away.

Further notes on reagents:

• Use 100 mL Coplin jar for microwave staining.
• Label all glassware as you make the solution to avoid confusion.
• The stock of 10% Gum mastic and 0.2% Gold Chloride is kept in fridge with 3 months shelf life.
• 2% Gold Chloride is reused, pour it back to stock after use.

Method:

1. Dewax (Twice xylene x 5 minutes, 100% alcohol x 3 minutes, 95% alcohol x 3 minutes, 70% alcohol x 3 minutes) sections and bring to water.
2. Make up the 10% Ammonium hydroxide and 1% Silver nitrate solution.
3. Wash in tap water for several minutes.
4. Rinse sections in distilled water for several minutes.
5. Place sections in a 100 mL Coplin jar.
6. Place sections in 10% Ammonium hydroxide for 10 minutes.
7. Rinse sections in distilled water for several minutes.
8. Make up the 0.5% Ammoniacal silver solution.
9. (Critical step!) Place sections in Ammoniacal silver nitrate in a Coplin jar with lid on. Heat in microwave in 5 second bursts (full power) for about 4 times, then agitate in 50°C water bath for 8-10 minutes (start with 6 minutes to avoid over-done) till section turns straw colour (check against white background). Dispose of silver nitrate solution in waste solution drum.
10. Rinse in distilled water thoroughly.
11. Get the 10% Gum mastic from fridge and make up 2.5% Gum mastic.
12. Rinse in 95% alcohol (make up in distilled water) thoroughly.
13. Rinse in 100% alcohol thoroughly.
14. Place in 2.5% Gum mastic in Coplin jar with lid on for 5 minutes.
15. Make up the 1.6% Hydroquinone and reducer.
16. Differentiate in reducer solution in microwave for 7 seconds.
17. (Critical step!) Further differentiate in reducer solution in 50°C water bath for approximately 2-3 minutes (start with 1 minute till the sections changed to chocolate colour). Go to step 14 immediately to stop the differentiation. Dispose solution after use.
18. Get the 0.2% Gold chloride from the fridge.
19. Rinse in 2 changes of 100% alcohol.
20. Tone section in 0.2% Gold chloride for 4 minutes until the sections turn grey (make sure grey colour developed evenly on the section).
21. Make up the 2% Sodium thiosulphate solution.
22. Rinse in distilled water.
23. Place sections in 2% Sodium thiosulphate solution for 1 minute (colour changed from grey to pink/purple).
24. Rinse in 4 changes of distilled water.
25. Drain Coplin jar and transfer slides to slide carriers.
26. Dehydrate (95% alcohol x 15 second, twice100% alcohol x 15 seconds and twice xylene x 15 seconds), clear and mount.

Results:

Axons – black

Dendrites – black

Neurofibrils – black

Neurofibrillary tangles – black

Plaques – black

Note: Sections which fail to brown adequately in Ammoniacal silver nitrate solution, can be re-exposed to fresh Ammoniacal silver nitrate for approximately 10 minutes with improved results.

Comments for consideration:

• The silver solution, type of water, freshness of the reagent and cleanliness of the glassware are critical areas to check to eliminate non-specific background staining.
• It is recommended to control the time slides are exposed to the gold chloride solution, as over toning causes obscuring of the plaques, neurofibrils and neurofibrillary tangles.
• As stated on the MSDS, small amounts of silver nitrate waste can be precipitated with salt (sodium chloride – NaCl) or carbonates and be reused. For larger amounts to be disposed of, the manufacturer should be contacted for additional information. Laboratory waste should be disposed of in accordance with the requirements of the local waste authority, the Environment Protection Authority and the Ministry of Health, as per appropriate National Standards (i.e. AS/NZS 2243, Parts2 to 5).

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.