Immunohistochemistry

Associate Professor Dr.

Özhan Eyigör

Uludag University College of Medicine

Department of Histology & Embryology

Advanced Research Techniques In Basic Medical Sciences

 Immunohistochemistry is a technique which is used

to demonstrate the antigens (most of the time the proteins) in cells and tissues

in their exact localization

by using a specific antigen-antibody reaction of which the antibody is labeled by many

different chemicals,

and

by preserving the structure of the cells and the tissues.

The History of the Immunohistochemistry

• 1941: Coons et all. have demonsrated antigens on tissue sections by using an antibody which was linked to an flousesans label.

• 1966: Nakane and Pierce as well as Avrameas and Uriel reported the use of secondary antibody which was conjugated with peroxidase enzyme.

• 1979: Sternberger described the peroxidase-antiperoxidase method.

• 1981: Hsu et all. have defined the method of avidin-biotin-peroxidase complex which is abbreviated as ABC method.

• Using microscopy and immunology together

• Highly specific binding of antibodies to their own antigens

• Making antibody-antigen complex microscopically visible

The Principles of Imuunohistochemistry

Where Immunohistochemistry is Used?

to detect:• Proteins, carbohidrates, nucleic acids, lipits• Types of the secreting cells • Membrane antigens• Structural antigens within the cytoplasm• Antigen localised in the nucleus

• Immunohistochemistry is frequently used both in experimental and diagnostical studies• Light, flouresans, confocal laser scanning or electron

microscope is used in order to visualize the labeled antibody-antigen complex.

Antibody

• ‘Y’ –shaped molecule in the structure of a glycoprotein. Both arms of the “Y” are termed “the binding parts” or “Fab fructures”. They are important in recognizing and binding the antigen.

• Two types of antibodies are used in immunohistochemistry• Polyclonal serum: contains a mixture of antibodies with

high binding affinity to different epitopes of an antigen. • Easy to produce and cheaper,• Raised in rabbit, guinea pig, goat or sheep,• Less specific than monoclonal antibodies.

• Monoclonal serum: contains one type of antigen which is very pure and specific to one epitope of an antigen.

• Difficult to produce or use, thus it is expensive,• Usually raised in mice,• Highly specific.

The characteristics of a good antibody:

1. High affinity to an antigen (affinity)

2. High binding property (avidity)

3. High concentration (titration)

The aim of the immunohistochemistry

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.

Antibody labelling methods:

1. Immunoenzyme method: An enzyme is used as the label. Peroxidase, alkalin phosfatase, glucose oxidaseChromogen (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 resultsLabeling intensity is lowUsed 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 longerMore sensitiveMore economic

Indirect method

Fluorescence Method

Texas Red,

Rodamin,

Cy3

AMCA

FITC,

Cy2

Immunolabelling methods:

3. Protein A method:

4. Unlabelled antibody methods: Enzym-antienzym method Peroxidase – antiperoxidase (PAP)Alkaline phosphatase - anti-Alkaline phosphatase

(APAAP)

Most sensitive resultsWidely usedApplied 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 sensitivityUsed 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 end-products, is used.:

• Peroxidase- hydrogen peroxide- diaminobenzidine (DAB):

• 3-amino-9-ethylcarbazole (AEC): • 4-chloro-1-naphthol (CN): KOYU MAVİ

BROWN

RED

DARK BLUE

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 -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, non-immune normal serum, gelatine, milk)

• Blocking the surface tension: (Tween 20, Triton X-100, NaCl)

• 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

Immunohistochemistry protocol -2

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.

• 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:

• 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

THANKS