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
| 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°C |
| 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 ※(注記) (modified) | 4g |
※(注記) 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 |