Protease 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.

The two enzymes used for protein digestion are trypsin and pepsin. Trypsin is used at a mildly alkaline pH, and pepsin in an acidic medium. Fixation may have a significant effect on the digestion. Formalin fixation should be minimal (no longer than overnight). Ethanol fixation, either as ethanol or as a mixture (Carnoy, for instance) is preferred.

Note: It is advisable to celloidinise sections after digestion as they can become quite friable and are likely to become detached from the slide.

Solutions

Trypsin			Pepsin
Phosphate buffer, 0.01M, pH 7.6	100 mL		Hydrochloric acid, 0.1N	100 mL
Sodium chloride	0.4 g		Pepsin powder	0.1 g
Sodium fluoride	0.1 g
Trypsin powder	0.1 g
Shake well for a few minutes. Filter before use.			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 the material to be removed.
2. Make sure they are hydrated.
3. Place one test section and one known positive section into the solvent used for the protease.
4. Place the other test section and the other known positive in the protease solution up to 18 hours at 22°C
5. Wash both digested sections gently with tap water
6. Celloidinise the sections if desired.
7. Stain all four sections together using the same procedure.
8. If the material 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 a carbohydrate-protein complex.
    

   Section	Undigested	Digested
   Known positive	If material is stained	And it is unstained
   Test sections	Then material stained	And unstained is a carbohydrate-protein complex

 

Reference

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