Principles of enzyme histochemistry

Enzyme histochemistry (also called catalytic histochemistry) is a method used to detect the activity of enzymes in the cells or tissues of an organism. Currently, about 150 enzymes have been demonstrated in situ, i.e. about 1/10 of the total number of enzymes. The very principle of catalytic activity consists of two steps.

The first step is the histochemical reaction itself: tissue with enzyme + substrate = product.

The second step is the "visualization reaction": a colored and insoluble compound is formed from the tested product of the first reaction.

In order for both of the above reactions to take place, certain conditions must be met.


 * Preservation of enzyme activity
 * Preservation of cell and tissue structure - cryostat sections (chemical fixation almost always reduces enzyme activity)
 * pH environment
 * Substrate in excess

We ensure these optimal conditions using an incubation environment that has the following components:


 * 1) Substrate: it should be soluble in water at a pH optimal for the given enzyme and should have the ability to create a colored final product, and at the same time penetrate quickly enough into the physiological localization of the enzyme. If the reaction product is colorless, a reagent used to make it visible is added to the IP.
 * 2) Activators: i.e. substances that increase enzyme activity
 * 3) Inhibitors: i.e. substances that reduce enzyme activity
 * 4) Suitable buffer: ensures the necessary pH optimum (phosphate, citrate, tris-maleate, acetate, etc.)

First, we offer the enzyme a suitable substrate:

Then we make the reaction product visible by reacting with a chromogen - visualization reaction :

The execution itself

 * The test is performed on free sections (on a glass slide or in tiny Petri dishes in an incubation solution) or on sections of adhesions stuck to a glass slide.
 * Incubate at 37°C or room temperature. The length of incubation is from a few minutes to two hours (depending on the amount of enzyme in the tissue and the sensitivity of the method)
 * Fixation:
 * fresh cryostat sections from unfixed tissue
 * sections from fixed specimens
 * sections prepared by lyophilization
 * Each histochemical certificate must be supplemented with a control experiment and inhibition tests.

Control experiment : serves to verify the results and is performed simply by omitting the substrate in the incubation solution.

Inhibition tests : are indicated in cases where the same substrate is attacked by several enzymes simultaneously.