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.

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 impregnation20% silver nitrate solution3 to 5 hours, 37℃
7RinsingDistilled waterQuickly
8Silver impregnationImpregnating solution16 to 20 hours, 37℃
9ReducingReducing solution10 minutes (reduce sections without washing in water)
10WashingRunning tap water 
11RinsingDistilled water 
12Toning0.2% gold chloride solution3 minutes
13WashingRunning tap water 
14Reducing2% oxalic acid solution5 minutes
15WashingRunning tap water 
16FixingFixing solution5 minutes
17WashingRunning tap waterMore than 10 minutes
18Dehydration70%, 95% ethanol5 minutes each
19Dehydration100%ehtanol3 changes, 5 minutes each
20ClearingXylene3 changes, 10 minuntes each

Impregnating solution

Boric acid-Borax buffer (pH8.2)20ml
0.5% silver nitrate solution1ml
10% pyridine solution1ml
distilled water100ml

Boric acid-Borax buffer recipe

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

0.2M boric acid solution

Anhydrous sodium sulfite５g
distilled water100ml

Issues to consider with staining