⑫ Staining

Holmes staining

The Holmes stain, which uses only inorganic stable reagents for silver staining of neurites, is a modification of Bodian's method.

The Holmes stain gives more consistant and reliable results and can be used as an alternative method to the Bodian stain.

1	Deparaffinization	Xylene	3 changes, 10 minuntes each
2	Removal of xylene	100% ethanol	3 changes, 5 minutes each
3	Hydration	95%, 70% ethanol	5 minutes each
4	Washing	Running tap water	2 minutes
5	Rinsing	Distilled water
6	Silver impregnation	20% silver nitrate solution	3 to 5 hours, 37℃
7	Rinsing	Distilled water	Quickly
8	Silver impregnation	Impregnating solution	16 to 20 hours, 37℃
9	Reducing	Reducing solution	10 minutes (reduce sections without washing in water)
10	Washing	Running tap water
11	Rinsing	Distilled water
12	Toning	0.2% gold chloride solution	3 minutes
13	Washing	Running tap water
14	Reducing	2% oxalic acid solution	5 minutes
15	Washing	Running tap water
16	Fixing	Fixing solution	5 minutes
17	Washing	Running tap water	More than 10 minutes
18	Dehydration	70%, 95% ethanol	5 minutes each
19	Dehydration	100%ehtanol	3 changes, 5 minutes each
20	Clearing	Xylene	3 changes, 10 minuntes each
21	Coverslipping

A 20 ml silver solution is required for 1 section (35 mm × 75 mm). Silver staining is more difficult when a small amount of silver solution is used.