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

Barrett's method for pituitary
Barrett, 1944.

• Dewax sections, rinse in alcohol, rinse in water.
• Stain with Weigert's haematoxylin  - 10 minutes.
• Wash in water.
• Differentiate in acid alcohol (1% hydrochloric acid in 70% alcohol) - 5 seconds.
• Wash in tap water - 5 minutes.
• Stain with fuchsin/ponceau mixture - 5 minutes.
• Wash off with 2% acetic acid.
• Stain with aniline blue - 5 minutes.
• Wash off with 2% acetic acid.
• Differentiate with red differentiator (30 seconds - 2 minutes).
• Dehydrate, clear and mount.

Results: 

Acidophil granules - red
Cytoplasm of chromophobe cells - pale blue/grey
Basophil granules - blue
Nuclei - blue/black

Fuchsin/ponceau
Acid fuchsin - 0.5g
Ponceau 2R - 0.5g
1% acetic acid - 100mls

Aniline blue
Aniline blue - 2.5g
Distilled water - 100mls
Glacial acetic acid - 2.5mls

Red differentiator stock
Phosphotungstic acid - 25g
Picric acid - 2.9g
95% alcohol - 100mls

Red differentiator
40 mls red differentiator stock
40mls 95% alcohol
20mls of distilled water

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