⑫ Staining

Holmes staining (Eguchi-Seki modification)

The Holmes stain is labor-intensive and time-consuming because the silver staining is performed twice. Moreover, the first of the two silver staining processes uses high-concentration silver nitrate aqueous solution (20%), which poses health and environmental concerns. Thus, we incorporated modifications to the Holmes staining method to enable sufficient staining without 20% silver nitrate aqueous solution.

• Staining method
• Impregnating solution
• Boric acid-Borax buffer recipe
• Reducing solution

Staining procedure

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

Impregnating solution

Boric acid-Borax buffer (pH8.2)	20ml
1% silver nitrate solution (modified)	３ml
10% pyridine solution (modified) >	６ml
distilled water	100ml
metallic copper ※ (modified)	４g

※ Cortex staining is improved by placing copper pieces in the silver solution. However, myelin sheaths become more noticeable at certain sites. In particular, for spinal cord cross-sections, it is better to avoid placing copper pieces in the silver solution.

Boric acid-Borax buffer recipe

pH	0.2M boric acid solution	0.5M boric acid solution
7.8	16	4
8.0	14	6
8.2	13	7
8.4	11	9
8.7	8	12
9.0	4	16

Reducing solution

Hydroquinone	1g
Anhydrous sodium sulfite	５g
distilled water	100ml