Tissue sample
5µ paraffin sections of formal sublimate fixed tissue is preferred.
Formalin fixed tissue is suitable.
Other fixatives are likely to be satisfactory.
Method
Bring sections to water via xylene and ethanol.
Stain nuclei to medium density with hemalum.
Wash in tap water for 5 minutes.
Blueing takes place in the phloxine solution.
Place in solution A for 20 minutes.
Rinse in tap water, blot almost dry.
Some technologists rinse with cellosolve instead of blotting.
The object is to remove all traces of water, as it
interferes with the ability of tartrazine to extract phloxine and counterstain.
Rinse with solution B to remove remaining water. Discard solution.
Place in solution B until inclusions are red and all other tissue is yellow.
The time varies considerably. Control microscopically.
Rinse thoroughly but briefly with absolute ethanol.
Do not rinse with water at this stage as it rapidly removes the
tartrazine. Some technologists use cellosolve instead of ethanol.
Clear with xylene and mount with a resinous medium.
Expected results
Nuclei – blue
Acidophil virus inclusion bodies – red
Paneth cell granules – red
Background – yellow
Notes
The 2-ethoxy ethanol must not be replaced by any other solvent.
The 2-ethoxy ethanol must be, and must remain, completely anhydrous.
Reference
Bancroft, J.D. and Stevens A. (1982) Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.
