Palmgren's Silver Impregnation
for Peripheral Nerves

Solutions

Solution A
Concentrated formalin	25	mL
Nitric acid, 1% aqu.	0.2	mL
Distilled water	75	mL

Solution B
Silver nitrate	15	g
Potassium nitrate	10	g
Glycine, 5% aqu.	1	mL
Distilled water	100	mL

Solution C
Pyrogallol	5	g
Nitric acid, 1% aqu.	1	mL
Absolute ethanol	275	mL
Distilled water	225	mL

Solution D
Gold chloride	1	g
Acetic acid, glacial	0.2	mL
Distilled water	200	mL

Solution E
Aniline	4	drops
Ethanol, absolute	100	mL
Distilled water	100	mL

Solution F
Sodium thiosulphate	5	g
Distilled water	100	mL

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.
Chrome, mercury and osmium fixatives should be avoided.

Method

1. Bring sections to water via xylene and ethanol.
2. Celloidinise sections.
3. Place into solution A for 15 minute at room temperature or for 5 minutes at 35°C.
4. Rinse with distilled water, three times over 5 minutes.
5. Do not rinse with water. Place into fresh solution C at 45°C for one minute. Agitate continuously.
6. Rinse with 50% ethanol.
7. Rinse thoroughly with distilled water, three times over 5 minutes.
8. Optionally, tone with solution D to the colour wanted.
9. Place into solution E for 15 seconds, agitating continuously.
10. Rinse with tap water.
11. Fix with solution F for 5 seconds.
12. Rinse well with tap water.
13. Dehydrate with ethanol, remove celloidin and clear with xylene. Mount with a resinous medium.

Expected results

• Nerve fibres  –  dark brown (untoned) or black (toned)
• Background  –  lighter brown (untoned) or grey (toned)

Notes

1. Toning with gold chloride (solution E) may be applied at step 8. Many workers find the black on grey scheme less satisfactory than dark brown on light brown.
2. If the nerve fibres are poorly shown, the impregnation may be intensified by repeating steps 2-7, but at lower temperature and shorter times. Toning and intensifying (steps 7-9) may also be repeated. These repetitions are often unsatisfactory.
3. If not celloidinised the preparations may have a coarse precipitate of silver.

 

Reference
Drury, R.A.B. and Wallington, E.A., (1980)
Carleton's histological technique Ed. 5
Oxford University Press, Oxford, UK.

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