Amylase Digestion

Enzymes can be used to remove materials from sections. The commonest of these is amylase (diastase) to remove glycogen. However, some carbohydrate-protein complexes can also be targeted with proteases, although this is much less common because they also digest the non-carbohydrate linked proteins comprising the vast majority of the tissue.

Diastase is available in many forms from many sources, and there has been considerable variation in the recommendations made over the years. Likely the earliest was the use of human saliva. This source is now strongly deprecated. Human pathogens are more likely to be found in unsterilised human materials, such as saliva, than in processed materials from nun-human sources, and the latter are far safer for the lab worker. Use of saliva is obsolete.

Amylases are available from both animal and plant sources. Hog pancreatic α-amylase is easliy obtained and highly recommended. Malt α- and β- amylases are also easily available, although Lillie points out they may contain ribonuclease (as does saliva). Many workers find the α-amylase superior to β-amylase for digestion of glycogen in fixed tissues. These enzymes are usually provided as powdered extracts and, like many biological products, are variable in their potency. They are often sold on the basis of the number of enzyme units available per milligram. Purchase products with a conveniently high activity when possible, and adjust the concentration of the digesting solution as needed.

Note: Celloidin acts as a physical barrier to amylase. If applied before digestion the treatment is unreliable. Celloidinise after digestion if wished.

Solution — Amylase

Chemical Amount
Malt or hog α-amylase 0.1 g
Phosphate buffer, 0.02M, pH 6.0 100 mL
Shake well for a few minutes.
Filter before use.

 

Procedure

  1. Prepare adjacent sections from the tissue to be tested and two sections known to contain glycogen.
  2. Make sure they are hydrated.
  3. Place one test section and one known positive section into water.
  4. Place the other test section and the other known positive in the amylase solution for 30-60 minutes at 35-45°C
  5. Wash both digested sections with tap water
  6. Celloidinise the sections if desired.
  7. Stain all four sections together using the same procedure.
  8. If the glycogen in the undigested known positive section is stained and the digested known positive section is unstained, then material stained in the undigested test section that is not present in the digested test section is presumed to be glycogen.
     
    Section Undigested Digested
    Known positive If glycogen is stained And it is unstained
    Test sections Then material stained And unstained is presumed to be glycogen

 

Reference

Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.

 


 

Translate in
Google Translate
Instructions