Laboratory Protocols

• Cryotomy
• Histological Staining Techniques
  • Index
• Histological fixatives
• Peroxidase-anti-peroxidase Technique
• Preparation of tissue section adhesives
• Processing tissues for light microscopy (basics) (PDF 1.1mb)
• Processing tissues for light microscopy (detailed)
• Sample collection guidelines
• Setting up the light microscope
• Specimen preparation for electron microscopy

Histological Staining Techniques

Luxol fast blue/cresyl violet

Kluver and Barrera, 1954.

Use 15µm sections.

• Take to 95% alcohol.
• Stain in luxol fast blue solution - 2 hours at 60^oC* or preferably overnight at 37^oC. (Use a screw-topped plastic coplin jar.)
• Wash in 95% alcohol.
• Rinse in distilled.
• Start differentiation by agitating for 10 seconds in a jar of 0.005% lithium carbonate. This solution begins to loose its differentiating ability after a few uses so replace it often with fresh.
• Differentiate for 30-60 seconds in 70% alcohol.
• Rinse all slides in distilled water and check the staining with a microscope.
• Repeat the three steps above until grey and white matter are clearly distinguishable. Nuclei should be colourless and myelin should stand out turquoise on a very pale grey/blue background.
• Stain in 0.1% cresyl violet in 1% acetic acid - 10 minutes.
• Wash in distilled.
• Rinse in 70% alcohol.
• Differentiate in 70% alcohol until only nuclei and nissl substance are purple - approx. 30 seconds.
• Rinse quickly in 100% alcohol, clear and mount.

(higher magnification)

Myelin - blue.
Red blood cells - turquoise.
Nuclei and nissl substance - purple.

* For the 2 hour timing use 0.05% lithium carbonate to differentiate instead of 0.005%, but be careful, it works much more quickly!

Luxol fast blue solution

Luxol fast blue MBS - 1g
95% alcohol - 1 litre
10% acetic acid - 5mls

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