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.
MEDICAL BIOLOGY Histochemistry and Cytochemistry LEC:4
2
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.
Note: the marker receive the ion that is splits from the substrate (e.g. phosphate from the phosphorylated molecules or oxygen from hydrogen peroxide). Thus the marker is not linked to the substrate but it introduce together with it.
Other examples of enzymes that can be detected histochemically include Phosphatases and Dehydrogenases:
Figure 1–10: Enzyme histochemistry, (a): Micrograph of cross sections of kidney tubules treated histochemically by the Gomori method for alkaline phosphatases show strong activity of this enzyme at the apical surfaces of the cells at the lumen of the tubules (arrows). (b): TEM image of a kidney cell in which acid phosphatase has been localized histochemically in three lysosomes (Ly) near the nucleus (N). The dark material within these structures is lead phosphate that precipitated in places with acid phosphatase activity. X25,00