MEDICAL BIOLOGY
Histochemistry and Cytochemistry
LEC:4
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 electrondense, 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.
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MEDICAL BIOLOGY
Histochemistry and Cytochemistry
LEC:4
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, electrondense 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.
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. X25,000.
Other examples of enzymes that can be detected histochemically include
Phosphatases and Dehydrogenases:
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MEDICAL BIOLOGY
Histochemistry and Cytochemistry
LEC:4
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.
Figure 4: Compounds that have affinity toward another molecule can be tagged with
a label and used to identify that molecule
Immunohistochemistry
A highly specific interaction between molecules is that between
an antigen and its antibody. For this reason, methods using labeled
antibodies have proved most useful in identifying and localizing specific
proteins and glycoproteins.
The body has cells that are able to distinguish its own molecules (self)
from foreign ones. When exposed to foreign molecules called antigens,
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MEDICAL BIOLOGY
Histochemistry and Cytochemistry
LEC:4
the body may respond by producing proteins antibodies that react
specifically and bind to the antigen, thus helping to eliminate the foreign
substance. In Immunohistochemistry, a tissue that may contain a certain
protein is incubated in a solution containing labeled antibody to this
protein which will binds to it and can be detected by microscopy.
For both diagnostic and research purposes, immunohistochemistry is very
widely used to detect specific proteins (or other molecules) of interest in
cells and tissues. This technique requires an antibody against the protein
that is to be detected, which means that the protein must have been
previously purified using biochemical or molecular approaches so that
antibodies against it can be produced. To produce antibodies against
protein x of a certain animal species (eg, a human or rat), the isolated
protein is injected into an animal of another species (eg, a rabbit or a
goat). If the protein's amino acid sequence is sufficiently different for this
animal to recognize it as foreign ( as an antigen) the animal will produce
antibodies against the protein.
H.W: Medical applications of immunocytochemistry.
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