⑫ Staining

Woelcke staining method for myelin sheath staining

Staining method that stains myelin sheaths with iron hematoxylin.

Staining procedure

1	Sectioning		15μm
2	Mounting sections		Affix to a silane-coated glass slide
3	Deparaffinization	Xylene	3 changes, 10 minuntes each
4	Removal of xylene	100% ethanol	3 changes, 5 minutes each
5	Hydration	95%, 70% ethanol	5 minutes each
6	Washing	Running tap water	2 minutes
7	Rinsing	Distilled water
8	Mordanting	2.5% ammonium iron(Ⅲ) sulfate solution	overnight
9	Washing	Distilled water	3 changes, 10 seconds each
10	Staining	Heidenhain's hematoxylin	1 to 4 hours
11	Washing	Distilled water	Several changes until the cortex turns white. It takes 1 to 2 hours
12	Dehydration	70%, 95% ethanol	5 minutes each
13	Dehydration	100% ehtanol	3 changes, 5 minutes each
14	Clearing	Xylene	3 changes, 10 minuntes each
15	Coverslipping

Staining solution

Heidenhain's hematoxylin

Prepare immediately before use.

Dissolve 1.5 g hematoxylin in 30 ml of 100% ethanol.
Add to 270 ml distilled water.
Add 15 ml saturated lithium carbonate.
Mix in order of 1 - 3.

Double staining with cresyl violet

Cresyl violet can be used as a counterstain in the Woelcke method
For frozen sections, this method is a particularly good alternative to KB staining.
Using KB staining for frozen sections can make non-specific responses more noticeable.

Photograph1：frozen section of cortex. Comparison of Woelcke staining (left) and KB staining (right)

Photograph2：Frozen section of frontal plane. Combining Woelche technic with Nissl stain

Photograph3：Frozen section of internal capsule. Combining Woelche technic with Nissl stain