Prussian Blue

This stain is for thick (1 cm) slabs of formalin fixed brain.

Solutions:

• Mulligan's Solution:
• 40 g Carbolic Acid (crystalline Phenol)
• 5 g Cupric Sulphate
• 1.25 ml conc. HCl
• 1000 ml distilled H₂O
• 1.0% ferric chloride (in 1 litre distilled H₂O
• 1.0% Potassium Ferrocyanide(in 1 litre distilled H₂O)

Staining Procedure

1. Heat Mulligan's Solution to 60° C in fumehood in a covered pyrex dish or loosely capped bottle.
2. Soak brain in distilled H₂O for 10 minutes
3. Pour Mulligan's Solution in a pyrex tray & gently place slabs in Solution for 4 minutes. Use spatulas. DO NOT TOUCH THIS SOLUTION
4. Transfer to tap water for 30-60 sec.
5. Transfer to 1% ferric chloride for 2 minutes.
6. Transfer to tap water for 3-5 minutes.
7. Transfer to 1% Potassium Ferrocyanide until the slabs begin to get dark blue, then remove them. The colour will intensify in the water rinse.
8. Wash for 5 min in gently running tap water.
9. Place in 70% Ethanol for preservation.

For Display:

Increase step 2 to 1-24 hours. In Step 8, rinse for 1 hr. and monitor before transferring to ethanol. Some water can de-stain sections.

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Steve Milway

Last modified: Wednesday, December 17, 2003