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

Goldner's Trichrome for Osteoid
Goldner

• Dewax sections, rinse in alcohol, rinse in water.
• Stain nuclei with Weigert's iron haematoxylin - 20 minutes.
• Wash in tap water - 1 minute.
• Differentiate in acid alcohol (1% hydrochloric acid in 70% alcohol) - not more than 5 seconds.
• Wash in tap water - 5 minutes.
• Rinse in distilled water.
• Stain in solution A for 5 minutes.
• Rinse with 1% aqueous acetic acid.
• Treat with solution B until collagen is decolourised. (Approx. 3 minutes.)
• Rinse with 1% aqueous acetic acid.
• Stain in solution C for 5 minutes.
• Treat with 1% aqueous acetic acid for 5 minutes.
• Blot dry, rinse quickly in 100% alcohol, clear and mount.

Results: 

Mineralised bone - green.
Osteoid - orange/red.
Nuclei blue - grey.
Cartilage - purple.

Solution A
Ponceau 2R - 0.075g
Acid fuchsin - 0.025g
Azophloxine - 0.01g
Acetic acid - 0.2ml
Distilled water - 100ml

Solution B
Orange G - 2g
Phosphomolybdic acid (dodeca-molybdophosphoric acid)- 4g
Distilled water - 100ml
Leave to stand for a couple of days for any precipitate to settle out and then filter before using.

Solution C
Light green - 0.2g
Acetic acid - 0.2ml
Distilled water - 100ml

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