Weil’s stain is a modification for paraffin sections of the Weigert-Pal-Kulschitsky technique. The underlying principle of these methods involves the reduction of chrome salt to chromium dioxide by myelin. The chromium subsequently acts as a mordant for the haematoxylin, intensifying the stain.

Procedure

This procedure is generally conducted on sections from formalin-fixed, paraffin-embedded tissue that are cut between 8-15 µm. Spinal cord tissue is rich in myelinated axons and can be used as a positive control.

1. Dewax and hydrate sections to distilled water.
2. Put slides in freshly prepared Staining Solution at 56-60C for 30 minutes.
3. Wash slides well in water.
4. Partially differentiate in iron alum differentiating solution until myelin sheaths stand out blueish-black on a pale grey background, approximately 5 minutes. If you are unsure, check your sections under the microscope at1 minute intervals).
5. Wash slides in tap water for 10 minutes.
6. Complete differentiation in Weigert’s differentiator, 1 to 2 minutes. Control this differentiation step carefully checking under the microscope, until the myelin is an intense deep blue  against a creamy or clear background.
7. Wash well in tap water.
8. Dehydrate through a series of graded ethanol baths, clear in xylene, and mount.

RESULTS

Myelin-containing structures will be stained black, red blood cells will be black, nuclei will be blue, and the background should be clear or yellow.

Solutions

Haematoxylin Solution, working strength

• 10g Haematoxylin
• 100 ml absolute ethanol.

Allow solution to “ripen” naturally for six (6) weeks, forming the basis of a stock solution. Just prior to staining, create a working strength solution by diluting the stock 1:4 with distilled water

Iron Alum Solution

• 4% (w/v) aqueous ferric ammonium sulphate.

Staining Solution

Mix equal volumes of preheated (56-60C) working strength Haematoxylin and Iron Alum solutions just prior to use.

Weigert’s Differentiator, 200 ml

• Borax (sodium tetraborate), 2g
• Potassium ferricyanide, 2.5g
• Distilled water, 200ml