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

Holmes’ method for axons

Holmes 1947
Use 10-20 micron paraffin wax sections.

• Dewax sections, rinse in alcohol, rinse in distilled water.
• Place in 20% silver nitrate in closed coplin jar - 2 hrs in the dark.
• In the meantime prepare the impregnating solution.
• Remove slides from the 20% silver nitrate solution and wash in 3 changes of distilled water over 10 minutes.
• Place the slides overnight in the impregnating solution at 37ºC in a sealed coplin jar.
• Shake off superfluous fluid and place the slides directly in the reducing solution - 2 minutes minimum. (10-15 mins recommended.)
• Wash in running tap water for 3 minutes then rinse in distilled water.
• Check slides microscopically for good contrast, return to reducing solution if insufficient.
• Tone in 0.2% gold chloride for 3 minutes.
• Rinse briefly in distilled water.
• Place in 2% oxalic acid  for 2-10 minutes, examine at intervals until the axons are blue-black colour. (4-5 mins recommended, too long may result in lower contrast.)
• Rinse in distilled water and fix with 5% sodium thiosulphate - 5 minutes.
• Wash in tap water - 10 minutes.
• Dehydrate, clear and mount.

Solutions:

Impregnating solution:

• 1.24% aqueous boric acid - 55ml
• 1.9% aqueous borax (Na2B4O7.10H2O) solution - 45ml
• Distilled water - 394ml
• 1% silver nitrate - 1ml
• 10% aqueous pyridine - 5ml
• Mix thoroughly

Reducing solution: 

• Hydroquinone 1g
• *Sodium sulphite 10g
• Distilled water 100 ml

Results:

(Axons)

(Muscle)

Axons - black
Background - greyish (turning purple with time)
Muscle striations - black

Notes:

Coplin jars should be thoroughly cleaned before use.

The 20% silver nitrate can be re-used but the impregnating must only be used once.

This method is applicable for both CNS and peripheral nerve material, and also demonstrates cross-striations of muscle fibres.

*When making up the reducing solution it is best to add the sodium sulphite to the water while it is being rapidly stirred. If not it tends to form a hard mass that is difficult to dissolve.

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