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

von Kossa, 1901
Use a positive control section.

• Dewax sections, rinse in alcohol, rinse several times in distilled water.
• Treat in 2% silver nitrate in a clear glass coplin jar placed directly in front of a bright lamp (see notes) - 1½ hours.
• Rinse in several changes of distilled water.
• Wash well in water.
• Rinse in distilled water.
• Counterstain with nuclear fast red - 1 minute.
• Rinse in distilled water.
• Dehydrate, clear and mount.

Results:

Ca++ - black.
Nuclei - red.

Notes:

• This laboratory uses a 100W tungsten-halogen projector lamp as the light source.  An ordinary 60-100W tungsten light bulb would suffice, although the time in the silver nitrate may have to be increased.
• Urates may also be blackened by this method.  If their presence is suspected a negative control may be prepared by treating a test section with a saturated solution of lithium carbonate for 5 minutes which will remove them.
• If there is any doubt that the resulting black deposits are calcium a negative control can be performed.  This is done by placing a second test section in 10% formic acid for 10 minutes prior to step 2, rinsing well in distilled water and continuing with the technique. Any calcium deposits are removed and the result is negative where the test slide is positive..

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