Histochemistry and Cytochemistry

Histochemistry and Cytochemistry
Objectives: Upon completion of this lecture, the student should be able to:
1- State the steps of enzyme histochemistry.
2- Explain the principle of Immunohistochemistry.

The terms histochemistry and cytochemistry indicate methods for localizing cellular structures in tissue sections using unique enzymatic activity present in those structures. To preserve these enzymes histochemical procedures are usually applied to unfixed or mildly fixed tissue, often sectioned on a cryostat to avoid adverse effects of heat and paraffin on enzymatic activity. Enzyme histochemistry usually works in the following way:
(1) Tissue sections are immersed in a solution that contains the substrate of the enzyme to be localized;
(2) The enzyme is allowed to act on its substrate;
(3) At this stage or later, the section is put in contact with a marker compound;
(4) This compound reacts with a molecule produced by enzymatic action on the substrate;
(5) The final reaction product, which must be insoluble and which is visible by light or electron microscopy only if it is colored or electron-dense, precipitates over the site that contains the enzyme. When examining such a section in the microscope, one can see the cell regions (or organelles) covered with a colored or electron-dense material.


Example:
Detection of Peroxidase enzyme: sections of adequately fixed tissue are incubated in a solution containing hydrogen peroxide and 3,3 -diamino-azobenzidine (DAB). The latter compound is oxidized in the presence of peroxidase, resulting in an insoluble, brown, electron-dense precipitate that permits the localization of peroxidase activity by light and electron microscopy.
Other examples of enzymes that can be detected histochemically include Phosphatases and Dehydrogenases:

Detection Methods Using Specific Interactions between Molecules
A molecule present in a tissue section may be identified by using compounds that specifically interact with the molecule. The compounds that will interact with the molecule must be tagged with a label that can be detected under the light or electron microscope (Figure 4). The most commonly used labels are fluorescent compounds (which can be seen with a fluorescence microscope), radioactive atoms (which can be detected with autoradiography), and metal (usually gold) particles that can be observed with light and electron microscopy. These methods are mainly used for detecting sugars, proteins, and nucleic acids.