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Module IB

What is histochemistry?
• It is a histological technique used for
Histochemistry studying chemistry of tissues and cells

Martin Špaček, MD • Histochemistry

(E-mail: m.spacek@centrum.cz) • Enzyme histochemistry
• Immunocytochemistry
• In situ hybridization


Histochemistry Histochemistry
Examples of Histochemical Methods: Examples of Histochemical Methods:
Ions Lipids

• Iron (ferric ions – Fe3+)

• stained with dyes soluble in the lipids
• Perls’s reaction:
– sections of tissues are incubated in a mixture of • eg. Sudan IV, Sudan black, Oil red, Nile
potassium ferrocyanide and hydrochloric acid blue
– result: insoluble dark blue precipitate of ferric • fresh frozen (cryostat) sections are used
for the most authentic picture of tissue
• Diagnostic application: lipids
– patients with diseases that store iron (eg.

Histochemistry Histochemistry
Examples of Histochemical Methods: Examples of Histochemical Methods:
Nucleic Acids – DNA Nucleic Acids – RNA

• Feulgen’s reaction: • RNA-rich organelles are stained with basic

– hydrolysis of DNA by hydrochloric acid dyes
– this process leads to the formation of • i.e.: toluidine blue, methylene blue
aldehyde groups • Since the RNA is not the only basophilic
– free aldehyde groups react with the substance in the tissue it is necessary to
Schiff reagent incubate a control slide with ribonuclease
– result: insoluble red substance

Histochemistry Examples of Histochemical Methods:
Examples of Histochemical Methods: 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)...

Enzyme Histochemistry Enzyme Histochemistry –

• Enzymes are the catalysts of most of • Peroxidase
biochemical reactions – sections are incubated in a solution containing
• Frozen tissues are used hydrogen peroxide and DAB
• Principle: – DAB is oxidized in the presence of peroxidase
– 1st reaction (histochemical):
– result: insoluble, black, electrondense precipitate
• enzyme + substrate → product
– 2nd reaction: • Clinical application:
• demonstration of the product – diagnosis of leukemias (detection of the
peroxidase activity in blood cells)
• Peroxidase is used also as a label in
immunocytochemistry and in ISH

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

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
• 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

Methods of Labelling Antibodies

• Enzyme labels (peroxidase)

• Colloidal metal labels (EM)
• Fluorescent labels
• Avidin-biotin techniques

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
• Fluorochromes: FITC (fluorescein
isothiocyanate), rhodamine, DAPI, etc.

Methods of Localizing Antigens In Situ Hybridization Techniques

• Direct method • Allow the demonstration of specific nucleic

– primary antibody is conjugated directly acid sequences in their cellular
to the label environment
• Are based on hybridization of a nucleic acid
• Indirect method (more sensitive)
probe (small lengths of nucleic acid of
– first step: nonlabeled anti-x antibody is known base sequence) to the target
bound to the antigen
• Applications:
– second: the labeled antiantibody binds – detection of abnormal genes
to the anti-x antibody – identification of viral infection, etc.

In Situ Hybridization Techniques

• Probes labelling:
– radioisotopes
– coupling of the nucleotides with biotin

– fluorescent labels (FISH)