Introduction
Enzyme Histochemistry
Enzyme histochemistry (also called cytochemistry) is a method for localizing cellular structures using a specific enzymatic activity present in those structures. This technique provides valuable insights into the functional aspects of tissues as enzymes play essential roles in various biochemical processes. For example, enzyme histochemistry can be used to study metabolic activity in various cell types.
To preserve enzymes for histochemical study, unfixed or mildly fixed tissues are sectioned on a cryostat to avoid the adverse effects of heat and organic solvents on enzymatic activity. This differs from the preparation of tissues for study by light microscopy where chemical fixatives like formalin are used to preserve tissue structure.
Here is a summary of the steps involved in enzyme histochemistry:
The first step is to immerse tissue sections in a solution containing the substrate of the enzyme to be localized.
The enzyme is allowed to act on its substrate.
The section is then put in contact with a marker compound that reacts with a product of the enzymatic action on the substrate.
The final product from the marker, which must be insoluble and visible by light or electron microscopy, precipitates over the site of the enzymes. This allows the region to be localized microscopically.
Some examples of the enzymes that can be detected histochemically include:
Phosphatases split the bond between a phosphate group and phosphorylated molecules. Alkaline phosphatases (those with maximum activity at an alkaline pH) and acid phosphatases can be detected. For example, alkaline phosphatase activity can be detected at the apical surfaces of cells at the lumens of kidney tubules.
Dehydrogenases remove hydrogen ions from one substrate and transfer them to another. They can be localized by incubating tissue sections in a substrate solution containing a molecule that receives hydrogen and precipitates as an insoluble colored compound. Mitochondria can be specifically identified by this method because dehydrogenases are among the citric acid (Krebs) cycle enzymes of this organelle.
Peroxidase promotes the oxidation of substrates with the transfer of hydrogen ions to hydrogen peroxide, forming water molecules. Peroxidase is commonly localized by histochemistry. For example, cell or tissue sections are incubated in a solution containing hydrogen peroxide and 3,3'-diamino-azobenzidine (DAB). DAB is oxidized in the presence of peroxidase to produce an insoluble, brown, electron-dense precipitate.