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

Puchtler, Sweat, Levine, 1962.
Cut sections at 6µm. Do not use gelatine coated slides. Use a positive control section.

• Dewax sections, rinse in alcohol, rinse in water.
• Stain in Harris haematoxylin - 10 minutes.
• Wash in tap water - 1 minute.
• Differentiate in acid alcohol - 10 seconds.
• Blue in tap water - 5 minutes.
• Treat with stock solution A - 20 minutes in a sealed coplin jar.
• Treat with stock solution B - 20 minutes in a sealed coplin jar.
• Rinse and dehydrate in 100% alcohol.
• Clear and mount.

Results: 

Amyloid, eosinophils, elastin - brick red. Amyloid also displays apple-green birefringence under polarized light.
Nuclei - blue.
Background - clear/pale pink.

Stock solution A
Saturated sodium chloride in 80% alcohol
Working solution = 50ml stock solution A + 0.5ml 1% aqueous sodium hydroxide

Stock Solution B
Saturated congo red in 80% alcohol saturated with sodium chloride
Working solution = 50ml stock solution B + 0.5ml 1% aqueous sodium hydroxide

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