PAS Reaction with Diastase Digestion

Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology (Third Edition). American Registry of Pathology ( Luna, Lee G., HT(ASCP) (editor)), McGraw Hill Publishers, New York 1960

Purpose
The demonstration of glycogen in tissue sections.
Principle
This is a very sensitive histochemical method for glycogen. Diastase and a-amylase act on glycogen to depolymerize it into smaller sugar units (maltose and glucose) that are washed out of the section. The Schiff reaction has been described in the PAS procedure.
Fixative
10% neutral buffered formalin, formalin alcohol, or absolute alcohol.
Equipment

* Hot plate pH meter
* Coplin jars
* balance
* Erlenmeyer flasks
* graduated cylinders
* filter paper

Technique
Cut two paraffin sections at 4 to 5 m. Label one section "with" and one section "without."
Quality Control
Two control sections of liver containing glycogen must be used, one labeled "with" and one labeled "without."
Reagents

* 0.5% Periodic Acid
* Periodic acid ..... 2.5g
* Distilled water ..... 500.0 ml
* IN Hydrochloric Acid
o Hydrochloric acid, concentrated (specific gravity, 1.19) ..... 83.5 mL
o Distilled water ..... 916.5 mL
Add the acid to the water and mix well.
* Schiff Reagent
o Distilled water ..... 800.0 mL
o Basic fuchsin ..... 4.0 g
o Sodiumetabisulfite ..... 4.0 g IN hydrochloric acid ..... 80.0 mL
Heat the water to the boiling point. Remove from flame, add the basic fuchsin, and reheat to the boiling point. Cool the solution to 50 C and Filter. Add 80.0 mL of IN HCI, cool completely, and add 4.0 g of sodium metabisulfite. Place the solution in the dark overnight. The solution should be light amber after standing. Add 2.0 g of activated charcoal and shake for I minute. Filter and store the solution in the refrigerator.
* Test for Quality of Schiff Reagent
(See the PAS procedure in this chapter.)
* 0.55% Potassium Metabisulfite
* Potassium metabisulfite ..... 2.75 g
* Distilled water ..... 500.0 mL
* Malt Diastase Solution
* Diastase of malt ..... 0.1 g
* Phosphate buffer, pH 6.0 ..... 100.0 mL
* Phosphate buffer, pH 6.0
* Sodium chloride ..... 8.0 g
* Sodium phosphate, monobasic . .. .. 1.97 g
* Distilled water . .. .. 100.0 mL
* Adjust pH to 6.0 if necessary.

Procedure

1. Deparaffinize and hydrate slides to distilled water.
2. Place the sections labeled "with" in preheated diastase solution at 37 C for I hour. Hold the sections labeled "without" in distilled water.
3. Wash in running water for 5 minutes.
4. Place all sections ("with" and "without") in 0.5% periodic acid solution for 5 minutes.
5. Wash in three changes of distilled water.
6. Place in Schiff reagent for 15 minutes.
7. Wash for 1 minute in each of two jars of 0.55% potassium metabisulfite to remove excess stain.
8. Wash in running tap water for 10 minutes to develop full color.
9. Counterstain minute in Harris's hematoxylin with acetic acid (2 mL acetic acid/48 mL hematoxylin).
10. Wash well in running water to blue the hematoxylin.
11. Dehydrate with two changes each of 95% and absolute alcohol, clear with xylene, and mount with synthetic resin.

Results
Glycogen will stain bright rose red on the section labeled "without" and will be absent from the section labeled "with."
Notes

1. Malt diastase, containing both a- and (3-amylase, is commonly used for digestion but tends to loosen the sections. For this reason as well as the decreased digestion time, many laboratories prefer to use human saliva, which contains only a-amylase. If preferred, digest with saliva for 20 minutes at room temperature.
2. Glycogen fixed in picric acid-containing fixatives may be more resistant to diastase digestion than when digestion follows other fixatives (Sheehan and Hrapchak).