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Techniques. Protocols


This protocol is intended to stain the myelin fo the central nervous system, but it is not useful for the peripheral nervous system. Woelcke staining makes visible the main axon tracts of the brain and the general nervous tissue organization, so that it can be used to detect alterations in neurophatology studies. In addition, it is also a good staining for detecting dead neurons, since they are labelled in black. Only the nucleus is stained in healthy neurons.


Samples are fixed in formaldehyde or Bouin and paraffin embedded. 8 µm thick sections are obtained and attached to gelatin coated slides.

1. 2x10 min in xylene

2. 2x10 min in 100º ethanol

3. 10 min in 96º ethanol

4. 10 min in 80º ethanol

5. 10 min in 50º ethanol

6. 5 min in distilled H2O

7. 16-18 h in 2.5 % of Ferric ammonium sulfate in distilled H2O
Ferric ammonium sulfate solution: 2.5 g of ferric ammonium sulfate in 100 ml of distilled H2O. The ferric ammonium sulfate crystals need to be pulverized and completely dissolved, and the color of the solution has to be purplish.

8. 1.5 a 2 h in hematoxylin working solution
(a) Alcoholic hematoxylin: 10 g hematoxylin in 100 ml of 100 º
(b) Lithium carbonate saturated solution: 1.45 g of lithium carbonate in 99 ml of distilled H2O
Hematoxylin working solution: 45 ml of distilled H2O + 10 ml of (a) + 7 ml of (b).
The hematoxylin working solution is prepared following this sequence. Try not to stir too much during the mixing of components. This solution can be used for about a week.

9. 80º ethanol untill the background of the tissue is clear. The clearing time depends on the staining intensity.

10. 5 min in 96º ethanol

11. 2x10 min in 100º ethanol

12. 2x10 min in xylene

13. Mounting medium


Nucleolus and glia: black

Myelin sheaths: dark blue

Degenerated neurons: black somata

Background: light grey


Try to prevent overstaining since it leads to dark backgrounds and myelin is hard to distinguish.

The time in ethanol series may be adjusted to get a proper staining, specially the 80º ethanol after the hematoxylin working solution.



50º, 70º, 80º, 90º, 96º y 100º ethanol

Hematoxylin (C.I. 75290)

Ferric ammonium sulfate

Lithium carbonate

Mounting medium


Staining dishes

Slides racks



Woelcke staining
Paraffin section, 8 µm, stained with Woelcke procedure. Myelin sheaths are clearly visible. Rat brain.
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