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 procedure

1DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95%, 70% ethanol5 minutes each
4WashingRunning tap water2 minutes
5RinsingDistilled water 
6Silver impregnationImpregnating solution16 to 20 hours, 37℃
7ReducingReducing solution10 minutes (reduce sections without washing in water)
8WashingRunning tap water 
9RinsingDistilled water 
10Toning0.2% gold chloride solution5 minutes
11WashingRunning tap water 
12Reducing2% oxalic acid solution5 minutes
13WashingRunning tap water 
14FixingFixing solution10 minutes
15WashingRunning tap waterMore than 10 minutes
16Dehydration70%, 95% ethanol5 minutes each
17Dehydration100% ehtanol3 changes, 5 minutes each
18ClearingXylene3 changes, 10 minuntes each

Impregnating solution

Boric acid-Borax buffer (pH8.2)20ml
1% silver nitrate solution (modified) 3ml
10% pyridine solution (modified) >6ml
distilled water100ml
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

pH0.2M boric acid solution0.5M boric acid solution

Reducing solution

Anhydrous sodium sulfite5g
distilled water100ml