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

Warthin, Starry, 1920. (Young's Modification)
Use organosilane treated slides. Pre-wash glassware with 10% nitric acid followed by de-ionised water. Use a positive control section. Use plastic forceps.

• Dewax sections, rinse in alcohol, rinse in water.
• Rinse in pH3.7 buffer.
• Prepare and heat solutions A & B to 56°C in a waterbath.
• Place slides in the 1% silver nitrate (solution A) at 56°C for 1 hour.
• 5 minutes before the hour is up add 2.5ml of 2% silver nitrate (solution C) to 45ml of gelatine (solution B) and pre-heat the quinol (solution D) in the waterbath.
• When the hour is up tip out the 1% silver (solution A) leaving the slides in the coplin jar.
• Quickly add 3ml of hydroquinone (solution D) to the silver/gelatin mixture and pour it into the coplin jar with the slides.
• Treat in this solution until the sections are golden brown. This can take 1-3 minutes.
• Wash carefully in warm tap water.
• Dehydrate, clear and mount.

Results:

Bacillus piliformis, spirochaetes and some other bacilli - black.
Nuclei and red blood cells - shades of brown.
Background - pale yellow.

Solutions:
A. 1% Silver Nitrate in buffer - prepare fresh each time do not use stock silver solutions.
    0.5g AgNO3 in 50ml pH3.7 buffer

B. 7% Gelatin solution
    3.1g gelatine in 45ml buffer
 
C. 2% Silver Nitrate in buffer - prepare fresh each time do not use stock silver solutions.
    0.2g AgNO3 in 10ml pH3.7 buffer

D. 4% Hydroquinone solution
    0.4g hydroquinone (quinol) in 10ml buffer

Acetate buffer stock solutions

• 0.2m sodium acetate (13.6g in 500mls de-ionised  water)
• 0.2m acetic acid (5.75ml in 500mls de-ionised  water)

Working pH3.7 buffer solution

• 0.2m sodium acetate 1.5ml
• 0.2m acetic acid 18.5ml
• De-ionised  water 480ml

Check the pH of the buffer before use.

Notes:

• The waterbath used for heating the solutions should be almost full so that the water level reaches far enough up the coplin jars to heat the solutions inside.
• The waterbath needs to be at approx. 58-60°C in order for the solutions to be at 56°C.
• Make sure all glassware is thoroughly cleaned before use (preferably with dilute nitric acid and several rinses of de-ionised  water.)
• Weigh all the chemicals very carefully as small discrepancies can affect the results.
• Be quick at the developing step (hydroquinone/silver/gelatin mixture) the solution doesn't last long once mixed.
• If the method fails suspect the hydroquinone first. This chemical deteriorates and the stain will not develop if it does. Check its age and replace with a new batch if the crystals looks brownish, they should be almost white.
  

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