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

Aldehyde fuchsin for pancreatic islet cells
(Use biopsied pancreas for control not PM material)
Halmi, 1952.

• Dewax sections, rinse in alcohol, rinse in water.
• Oxidise with Lugol's iodine - 10 minutes.
• Rinse in tap water and decolourise in 2.5% sodium thiosulphate.
• Wash well in tap water.
• Rinse in 70% alcohol.
• Stain with aldehyde fuchsin solution in a closed coplin jar for 15 - 30 minutes.
• Rinse in 95% alcohol.
• Rinse in tap water.
• Stain with celestine blue - 5 minutes.
• Rinse in tap water.
• Stain with Harris' haematoxylin - 5 minutes.
• Wash in tap water - 1 minute.
• Differentiate in acid alcohol (1% HCl in 70% alcohol) - 10 seconds.
• Blue in tap water - 5 minutes.
• Counterstain in orange G/light green - 45 seconds.
• Rinse in 0.2% acetic acid followed by 95% alcohol.
• Dehydrate, clear and mount.

Results:

Endocrine cells: A cells - yellow, B cells - purple, D cells - green.
Collagen - green.
Elastin and mast cells - violet.
Nuclei - black/brown.

Aldehyde fuchsin
Pararosaniline, colour index number C.I. 42500 - 1g
Paraldehyde (Paracetaldehyde) - 2mls
Conc. HCl - 1ml
Ethanol - 60mls
Distilled water - 40mls
Dissolve the pararosaniline in the alcohol and distilled water mixed. Add the HCl and the paraldehyde. Allow to ripen for 2 - 7 days at room temperature then filter. Store at 4^oC. (Under-ripened solution will show non-specific purple staining.)

Orange G/ light green
Distilled water - 100mls
Phosphotungstic acid - 0.5g (dissolve before adding the dyes)
Light green - 0.2g
Orange G - 1g
Glacial acetic acid - 1ml

Celestine blue
Celestine blue B - 0.5g
Ferric ammonium sulphate (iron alum) - 5g
Glycerin - 14ml
Distilled water - 100ml
Dissolve the iron alum in the distilled water without heat.  Add the celestine blue and boil for 3 minutes.  Filter when cool and add the glycerin.  The stain should keep for at least 6 months.

 

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