⑫ Staining

Woelcke staining method for myelin sheath staining

Staining method that stains myelin sheaths with iron hematoxylin.

Staining procedure

1Sectioning 15μm
2Mounting sections Affix to a silane-coated glass slide
3DeparaffinizationXylene3 changes, 10 minuntes each
4Removal of xylene100% ethanol3 changes, 5 minutes each
5Hydration95%, 70% ethanol5 minutes each
6WashingRunning tap water2 minutes
7RinsingDistilled water 
8Mordanting2.5% ammonium iron(Ⅲ) sulfate solutionovernight
9WashingDistilled water3 changes, 10 seconds each
10StainingHeidenhain's hematoxylin1 to 4 hours
11WashingDistilled waterSeveral changes until the cortex turns white. It takes 1 to 2 hours
12Dehydration70%, 95% ethanol5 minutes each
13Dehydration100% ehtanol3 changes, 5 minutes each
14ClearingXylene3 changes, 10 minuntes each

Staining solution

Heidenhain's hematoxylin

Prepare immediately before use.

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

Double staining with cresyl violet

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