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What is histochemistry?
• It is a histological technique used for
Histochemistry studying chemistry of tissues and cells
http://www.lf3.cuni.cz/histologie
Histochemistry Histochemistry
Examples of Histochemical Methods: Examples of Histochemical Methods:
Ions Lipids
Histochemistry Histochemistry
Examples of Histochemical Methods: Examples of Histochemical Methods:
Nucleic Acids – DNA Nucleic Acids – RNA
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Histochemistry
Histochemistry Examples of Histochemical Methods:
Examples of Histochemical Methods: Saccharides
Saccharides
• PAS-positive substances are:
– polysaccharides (glycogen)
• Saccharides can be detect by PAS
– glycosaminoglycans /mucopolysaccharides/
reaction (periodic acid-Schiff)
(hyaluronic acid, chondroitin sulphate)
• it is based on oxidative action of periodic – proteoglycans
acid (HIO4) → aldehyde groups – glycoproteins (thyreoglobulin, collagen)
• these aldehyde groups react with Schiff’s – glycolipids (lipofuscin)
reagent (as in Feulgen’s reaction) • Clinical application:
• → a new compound with a purple colour – biopsies of tissues from patients with diseases
(PAS-positive substances) that store glycogen (glycogenosis),
glycosaminoglycans (mucopolysaccharidosis)...
Immunocytochemistry Antibodies
• Is a technique for identifying cellular or An antibody
tissue constituents (antigens) by means of molecule is
antigen-antibody interactions composed of four
• Antigens: polypeptide chains,
– proteins, glycoproteins, proteoglycans two identical light
• Antibodies: chains and two
– serum proteins known as immunoglobulins identical heavy
– formed in the humoral immune system by chains
plasma cells
– there are five types of antibody found in the
blood IgA, IgD, IgE, IgG and IgM.
– IgG is the commonest and the most frequently
used antibody for immunohistochemistry
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Antigenic Determinants (Epitopes) Monoclonal antibodies
• The specific site of an antigen that binds to an • Monoclonal antibodies are “cloned” antibodies that all
antibody is called an antigenic determinant or respond to exactly the same epitope of the same
epitope antigen – they are exceptionally specific
• Most antigens have a variety of epitopes that • Activated B cells (plasma cells) contain a mixture of
antibodies directed against different epitopes
generate a number of different antibodies that are
called polyclonal • Activated B cells have a limited lifetime when grown in
culture
• A single immune response to an antigen is termed • How can we select a single B cell clone (monoclonal)
monoclonal and propagate the cell line?
Monoclonal antibodies
Monoclonal antibodies
• B lymphocytes can
• Antibodies can be raised in mice by injection mutate into tumor cells
of an antigen. that result in a type of
• Repeated injections create a pool of activated cancer termed
B cells. myeloma.
• Myeloma cells become
“immortal” and will grow
indefinitely in culture.
• Fusion of a single
activated B cell and a
myeloma cell will create
a hybridoma that can
grow indefinitely in
culture
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Fluorescent Compounds Avidin-biotin Techniques
• Fluochromes are substances which can
• These methods rely on the affinity of the
absorb radiation in the form of ultraviolet or
glycoprotein avidin for biotin
visible light
• Biotin, a low molecular weight vitamin, is
• This absorbed radiation causes an “excited
easily conjugated to antibodies and
state” followed by emission of radiation of
enzyme markers
a different wavelength
• Up to 150 biotin molecules can be attached
• The fluorescent substances then appear
to one antibody molecule → increased
as shiny particles on a dark background
sensitivity
• Fluorochromes: FITC (fluorescein
isothiocyanate), rhodamine, DAPI, etc.