PEARSE' PAS Orange G
for Pituitary α & β cells
Materials
- Periodic acid – 1% aqueous
- Schiff’s reagent
- Celestine blue
- Mayer’s hemalum
- Orange G
Material Amount Orange G 4 g Phosphomolybdic acid 10 g Distilled water 200 mL
Preparation
- Dissolve the dye and acid in the water by shaking periodically for a few days, then leave completely undisturbed until the solution clarifies.
- Pipette off the clear solution and store in a tightly stoppered bottle. Do not filter to clarify the solution.
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Mercuric chloride fixation was originally recommended. Other fixatives are usually satisfactory, but glutaraldehyde should be avoided.
Protocol
- Bring sections to water via xylene and ethanol.
- Place into periodic acid for 10 minutes.
- Rinse well with tap water.
- Rinse with distilled water.
- Place in Schiff’s reagent for 10-20 minutes.
- Wash off with distilled water.
- Wash well with tap water for about 10 minutes.
- Counterstain with the celestine blue-hemalum sequence,
- Wash well with tap water for 5 minutes.
- Place in orange G for 20 seconds.
- Rinse with tap water until pale orange or check microscopically.
- Dehydrate with ethanol, clear with xylene and coverslip using a resinous medium.
Expected Results
- Erythrocytes – yellow
- Oxidisable carbohydrates – red
- Nuclei – blue
- Pituitary α cells – orange
- Pituitary β cells – red
- Pituitary chromophobe cells – unstained
Notes
- Lillie recommended Orange II instead of Orange G as it gave a deeper orange colour.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Lillie, R. D. and Fullmer, H.M., (1990)
Histopathologic technique and practical histochemistry, Ed. 4, pp. 278
Churchill Livingstone, London, England. - Bancroft, J. D. & Stevens, A., (1990)
Theory and practice of histological techniques, Ed. 3, pp. 345
McGraw-Hill Book Co., New York, USA.
