Lillie's Short PAS Reaction
for 1-2 Glycols
The periodic acid Schiff reaction (PAS) is used to demonstrate the presence of 1-2-glycols, and is consequently an important method in the histochemistry of carbohydrates and the histological demonstration of many structures.
Lillie referred to this method as
The Periodic acid, Schiff Sulfite Leucofuchsin Reaction, short variant.
Materials
- Periodic acid (potassium iodate, nitric acid specified).
- Schiff’s reagent.
- Mayer’s hemalum, or Weigert’s iron hematoxylin and saturated aqueous picric acid.
- Sodium metabisulfite, 0.5% aqueous
- Pal’s bleach
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory, although glutaraldehyde should be avoided.
Protocol
- Bring sections to water via xylene and ethanol.
- Oxidize in periodic acid for ten minutes.
- Wash in running water for five minutes.
- Place in Schiff’s reagent for 10 minutes.
- Transfer to three successive baths of sodium metabisulfite for 1, 2, and 2 minutes respectively.
- Wash in running tap water for 5 minutes.
- Counterstain with one of the following:
- Place in Mayer’s hemalum for 2 minutes. Wash in running tap water and blue.
- Place in Weigert’s iron hematoxylin for 2-4 minutes. Decolorise with Pal’s bleach diluted 1:5 with distilled water. Wash in running tap water for 4 minutes.
- Place in Weigert’s iron hematoxylin for 6 minutes. Wash for 4 minutes in running tap water. Place in saturated aqueous picric acid for 1 minute.
- Dehydrate with ethanol, clear with xylene, and coverslip using a resinous medium.
Expected Results
- 1-2-glycols – red
- Nuclei – blue or black
- Cytoplasm – yellow or unstained
Notes
- The nuclear counterstain may obscure some positive staining. Keep the application time short.
- Weigert’s solution should be allowed to ripen for an hour before use.
- Weigert’s hematoxylin and Pal’s bleach combination gives highly selective nuclear staining.
- Picric acid differentiates the nuclear stain as well as coloring the background.
- Glutaraldehyde fixed tissues will have a non-specific positive background staining. This must be blocked before step 2.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.
