Immunohistochemistry
Aulanni’am
University of Brawijaya
Labelling_Conjugated
Where Immunohistochemistry is Used?
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to detect:
Proteins, carbohidrates, nucleic acids, lipits
Types of the secreting cells
Membrane antigens
Structural antigens within the cytoplasm
Antigen localised in the nucleus
The aim of the immunohistochemistry
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to perform most specific immunohistochemical
staining;
by cousing least damage on the cell or tissue,
by using least amount of antibody,
in shortest time,
with least backgroung staining.
Monoclonal & Polyclonal
Polyclonal
Monoclonal
multi antigenic sites
One antigenic site
Various classes of
antibodies are present
(IgG,IgM,and so on)
Single class of antibody
produced
Can make a specific
antibody using only
a highly purified antigen
Can make a specific
antibody using an
impure antigen
Highly reproducible
Reproducibility and
standardization difficult
Antibody labelling methods:
1. Immunoenzyme method: An enzyme is used as the label.
Peroxidase, alkalin phosfatase, glucose oxidase
Chromogen (staining chemical) must be used
2. Immunofluorescence method: A fluorochrome is used as
the label.
AMCA, Fluorescein, FITC, Rodomine, TRITC, Texas Red.
Fluorescence microscope with appropriate filter or confocal
laser scanning microscope must be used to visualize.
3. Immunogold method: colloidal gold particles are used as
the label.
Usually used in electron microscopy.
Immunolabelling methods:
1. Direct method: The antigen directly binds to its specific
labelled antibody (primary antibody)
Fast to get the results
Labeling intensity is low
Used for kidney or skin biopsies.
Direct method
Immunolabelling methods:
2. Indirect method: Primary antibody is unlabelled. A
secondary antibody (which is labeled) is used.
Sekondary antibody must be raised against the immunoglobulin of the
species which the primary antibody is made in.
Getting results takes longer
More sensitive
More economic
Indirect method
Fluorescence Method
Texas Red,
Rodamin,
Cy3
FITC,
Cy2
AMCA
Immunolabelling methods:
3. Protein A method:
4. Unlabelled antibody methods:
Enzym-antienzym method
Peroxidase – antiperoxidase (PAP)
Alkaline phosphatase - anti-Alkaline phosphatase (APAAP)
Most sensitive results
Widely used
Applied on paraffin, cryostat sections or on smears.
Peroxidase –
antiperoxidase
(PAP)
Alkaline phosphatase anti-Alkaline
phosphatase (APAAP)
Immunolabelling methods:
5. Avidin-Biotin method:
Uses the high affinity of Avidin (glycoprotein) for biotin
(vitamin).
A complex of avidin-biotin-enzym (peroksidaz) is
necessary.
Streptoavidin can be used instead of avidin.
The secondary antibody is labelled with biotin which
inturn binds to avidin in the avidin-biotin-enzym complex.
Very high sensitivity
Used in research more than routine studies. It is longer and
more expensive.
• In order to visualize the enzymes labelling the antibodies
with light microscope, enzyme – substrate reactions, which
convert colorless chromogens into visible colored endproducts, is used.:
• Peroxidase- hydrogen peroxide- diaminobenzidine
(DAB): BROWN
• 3-amino-9-ethylcarbazole (AEC): RED
• 4-chloro-1-naphthol (CN): KOYU
DARK MAVİ
BLUE
Radioisotope labels
 Advantages
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Flexibility
Sensitivity
Size
 Disadvantages
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Toxicity
Shelf life
Disposal costs
Enzyme labels
 Advantages
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Diversity
Amplification
Versatility
 Disadvantages
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Lability
Size
Heterogeneity
Fluorescent labels
 Advantages
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Size
Specificity
Sensitivity
 Disadvantages
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Hardware
Limited selection
Background
IgG
IgG
Confocal
Application in renal diseases
IgG
The basic principle of
immunofluorescence
 To
use a fluorescent compound (usually
fluorescein) to detect the binding of antigen and
antibody
 The Ab is labelled with the fluorescent compound
 Under a fluorescence microscope, fluorescein
appears bright green wherever the binding occurs
Green fluorescence of FITC
Using the fluorescence microscope
 Select the correct filter block for the fluorescent
compound
 Fluorescence fades quickly under UV light; try to
limit the time of exposure to UV as much as possible
 Use high speed films for photography
Direct Immunofluorescence
 The aim is to identify the presence and location of an
antigen by the use of a fluorescent labelled specific
antibody
One step
Direct Immunofluorescence
Two step
Direct Immunofluorescence
Medical applications of direct IF
 Renal diseases for evidence of immune deposition
 Skin diseases for evidence of immune deposition
 Detection of specific antigens, especially those of
infective organisms
Application in renal diseases
IgG
 A section of kidney is placed on a slide; a
fluorescein-labeled antiglobulin (specific for
IgG, in this case) is added, then rinsed away
 The presence of fluorescence in the glomeruli
indicates that IgG was deposited prior to the
biopsy
 IgG is deposited in granular clumps along the
capillary walls, enabling a diagnosis of
membranous glomerulonephritis in this case
Chemiluminescent labels
 Advantages
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Size
Sensitivity
S/N
 Disadvantages
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Hardware
ELISA
The ELISA technique is used widely to detect
and quantitate organisms and/or their products in
foods, and synopses of some of these applications are
presented below. For more details, the cited
references should be consulted.
Enzyme-linked immunosorbent assay
Substrate
2nd antibody
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Specimen
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Microtiter well
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ELISA (variation 1)
Specimen
Labeled antigen
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Microtiter well
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ELISA (variation 2)
Labeled antibody
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Specimen
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Microtiter well
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Immunohistochemistry protocol -1
• Before incubation with antibody:
• Deparaffinization.
• Removing the fixative: washing in buffer solutions
(phosphate buffer, Tris-HCl buffer, HEPES buffer, etc)
• Neutralization of the endogeneus peroxidase
• Blocking: covering the non-immunological sticky
sites on tissues (bovine serum albumine, nonimmune normal serum, gelatine, milk)
• Blocking the surface tension: (Tween 20, Triton X-100,
NaCl)
Tissue Preparation
• Fixation: Immersion or perfusion fixation
• Neutral formaline
• Paraformaldehyde
• Paraformaldehyde ve picric acid (Bouin’s solution)
• Sectioning:
• Microtome: paraffin blocks
• Cryostat: frozen tissue
• Vibratome: fixed hard tissue
• Sections on a slide (PAP Pen)
• Floating sections
Immunohistochemistry protocol -2
• Incubation:
Primary antibody: Used in a solution at different dilutions with
the blocking agent. The incubation time changes according to
the properties of the antigen or antibody as well as depending
on the temperature.
Secondary antibody: Must be raised in the species other
than the species of which the cells or tissues are taken or
the primary antibody is raised. It should specifically
recognize the immunoglobuline of the species in which the
primary antibody is raised.
• Labelling: Immunoenzyme, immunoflourescence, immunogold
• Microscobical analyses
Determining the Secretory
Contents of the
Neuroendocrine Neurones
Multiple immunolabelling
• Detecting more than one antigen in the same cell or on the same
tissue.
• A combination of different single labelling methods is used.
• Cross-reaction must be avoided:
• Using primary antibodies raised in different species.
(not necessary if antigens of interest are localized in different
compartments of the cell such as cytoplasm vs nucleus.)
• The secondary antibodies must be raised in the same species
such as donkey.
• Specificity of the primary antibodies must be controled before.
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Light Microscobe (two antigens, sometimes three)
Fluorescence (two or three) or confokal microscobe (two-five)
Combining two techniques (two-six)
Electron microscobe (usually two)
GnRH and P-CREB
The use of the Immunohistochemistry:
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Intercellular antigens, for instance: immunoglobulines of
the kidney glomerular basal membrane
Cell surface antigens, tissue antigen for diagnosing
autoimmune diseases
Protein hormones in histopathological diagnosis
Soluble antigens of the cell
Diagnosis of the endocrine tumors
Small amounts of peptides in endocrine or neuroendocrine
cells
Immunodeposits
Tumoral markers
Tumor typing
Double labelling
Indirect Immunofluorescence
Indirect Immunofluorescence
 The aim is to identify the presence of antigen specific
antibodies in serum. The method is also be used to
compare concentration of the antibodies in sera.
Indirect Immunofluorescence
 A known antigen is placed on a slide; the patient's
serum is added, then rinsed away.
 A fluorescein-labeled antiglobulin is added, then
rinsed away.
 The presence of fluorescence over the antigen
indicates the presence of antibodies to this antigen in
the patient.
Diagnosis of Bacterial Diseases
 Clostridial diseases (direct)
 Brucella canis (indirect)
 Afipia catei, cat scratch disease (indirect)
 Borrelia burgdorferi (indirect)
 Coxiella burnetii, Q Fever (indirect)
 Rickettsia rickettsiae, Rocky Mountain Spotted
Fever (indirect)
Diagnosis of Viral Diseases
 rabies virus (direct)
 bovine immunodeficiency-like virus (indirect)
 canine coronavirus (indirect)
 canine distemper (indirect)
 feline infectious peritonitis (corona-) virus
(direct)
 porcine respiratory and reproductive syndrome
(indirect)
Diagnosis of Protozoal Diseases
 Babesia species (indirect)
 Ehrlichia species (indirect)
 Toxoplasma gondii (indirect)
 Trypanosoma cruzi (indirect)
 Cryptosporidia/Giardia (direct)
 Encephalitozoon cuniculi (indirect)
 Neosporum caninum (direct, indirect)
Some examples
INDIRECT IMMUNOFLUORESCENCE
for Antibodies to Toxoplasma
gondii
for Antibodies to Toxoplasma
gondii
 Toxoplasma organisms are killed and placed on the slide;
the patient’s serum is added, then washed away.
 A fluorescein-labeled antiglobulin is added, then washed
away.
 The presence of the green fluorescence outlining the T.
gondii organisms indicates the presence of antibodies in the
patient's serum.
for Antibodies to Toxoplasma
gondii
Immune-Mediated Disorders
 antinuclear antibody (ANA) test (for diagnosis of
systemic lupus erythematosus)
 Direct fluorescent antibody test for deposition of Abs
in tissues, e.g. kidney, skin
Indirect Fluorescent Antibody
Test for Antinuclear Antibodies
Indirect Fluorescent Antibody
Test for Antinuclear Antibodies
 Cells from a cultured cell line are placed on a
slide; the patient's serum is added, then rinsed
away.
 A fluorescein-labeled antiglobulin is added,
then rinsed away.
 The presence of fluorescence in the nucleus of
these cells indicates the presence of antibodies
to nuclear antigens in the patient.
Indirect Fluorescent Antibody
Test for Antinuclear Antibodies
Advantage over Immunoperoxidase
 Technically easier (fewer steps)
 More sensitive results
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