⑫ Staining

Marchi staining method for denatured myelin sheath staining

Only denatured myelin sheaths have reduced osmium tetroxide and stain as black granules.

Staining procedure

1	Dissecting a fixed brain		2 to 3mm
2	Staining	Staining solution	Place tissues in a sealable container and shake. Leave for 7- 21 days
3	Washing	Running tap water	1 day
4	Dehydration		*See Dehydration
5	Clearing		*See Clearing
6	Paraffin impregnation		*See Paraffin impregnation
7	Paraffin embedding		*See Paraffin embedding
8	Sectioning		*See Sectioning
9	Deparaffinization	Xylene	3 changes, 10 minuntes each
10	Coverslipping

Note

The entire section will turn black if it is left in ethanol for a long period.
The myelin sheaths will be stain from approximately 8 days after the nerves were damaged.

Staining solution

1% potassium chlorate solution	60ml
1% osmium tetroxide solution	20ml
acetic acid	1ml
37% formalin solution	12ml