Lillie's Long PAS
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 as
"The Periodic acid Leucofuchsin Method in Relation to Various Modifying Procedures".He describes it's purpose as being
"To indicate at what points and in what sequence various modifying procedures should be introduced".
A periodic acid solution.
A strong Schiff's reagent.
Sodium metabisulphite, 0.5% aqueous, optional.
Mayer's hemalum, or Weigert's iron hematoxylin.
Picric acid, saturated aqueous, optional.
Orange G, 2% aqueous, optional.
Methyl blue, 0.1% in saturated aqueous picric acid, optional.
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory, although glutaraldehyde should be avoided.
|Acetylation||Acetylation blocks hydroxyl and amino groups. Cartilage and glycogen are the most resistant. Other carbohydrates are more easily affected.|
|Benzoylation||Similar to acetylation, this also blocks hydroxyl and amino groups. Again, cartilage and glycogen are the most resistant while other carbohydrates are more easily affected.|
|Deacetylation (saponification)||Deacetylation (also called saponification) reverses the effects of acetylation in some, if not all of the materials which were blocked. The reversal is progressive, that is, some substances recover their stainabilty before others.|
|Methylation||Extended methylation causes some otherwise oxidisable carbohydrates to become non-reactive. Primarily, however, it inhibits basophilia.|
|Aldehyde block||If applied before periodic acid oxidation, pre-existing aldehydes are inhibited from reacting and will be unstained. If applied after periodic acid oxidation, aldehydes produced by the periodic acid are inhibited from reacting and will be unstained. A second section which has not been blocked can be used to prove that oxidation formed the aldehydes.|
|Amylase digestion||Amylase (diastase) is used to remove glycogen. This is either to prove that a subtance is glycogen, or to remove it so that other PAS positive materials other than glycogen may be more clearly seen.|
|Protease digestion||Proteases may also be used to remove carbohydrate-protein complexes, although this is less common than amylase digestion because the enzymes also digest other tissue proteins.|
|Reducing rinses||This is an acidified solution of potassium iodide and sodium thiosulphate applied following periodic acid to remove any iodate or periodate remaining in the tissue.|
& Solvent extraction
|PAS positive carbohydrate-lipid complexes, or glycolipids, may be removed with these solvents. They may be used at ambient or elevated temperature.|
Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.