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

Alizarin red S for calcium
McGee-Russell, 1958.
Use a positive control section.

• Dewax sections, rinse in alcohol, rinse in water.
• Rinse in distilled.
• Filter on alizarin red solution and leave for - 1 minute.
• Blot dry then rinse in acetone - 30 seconds.
• Rinse in acetone/Histo-Clear^® (or acetone/xylene) 1:1 - 15 seconds.
• Rinse in Histo-Clear (or xylene) and mount.

 

Results

:
Calcium deposits - orange/red.

Alizarin red solution
Alizarin red S- 2g
Distilled water - 100mls

Adjust pH to approx. 4.2 with 10% hydrochloric acid then with 10% ammonium hydroxide if it goes below.

If there is any doubt that the resulting red staining is calcium a negative control (to remove calcium) can be performed.  This is done by placing a second test section in 10% formic acid for 10 minutes prior to step 3, rinsing well in distilled water and continuing with the technique.

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