1. Immunohistochemistry
Advanced Research Techniques In Basic Medical Sciences
Immunohistochemistry
Associate Professor Dr.
Özhan Eyigör
Uludag University College of Medicine
Department of Histology & Embryology

2.  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.

3. 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.

4. The Principles of Imuunohistochemistry
Using microscopy and immunology together
Highly specific binding of antibodies to their own antigens
Making antibody-antigen complex microscopically visible

5. 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.

6. 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.

7. The characteristics of a good antibody:
High affinity to an antigen (affinity)
High binding property (avidity)
High concentration (titration)

8. 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.

9. Antibody labelling methods:
Immunoenzyme method: An enzyme is used as the label.
Peroxidase, alkalin phosfatase, glucose oxidase
Chromogen (staining chemical) must be used
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.

10. Immunolabelling methods:
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.

11. Direct method

12. Immunolabelling methods:
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

13. Indirect method

14. Fluorescence Method
Texas Red,
Rodamin,
Cy3
FITC,
Cy2
AMCA

15. Immunolabelling methods:
Protein A method:
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.

16. Peroxidase – antiperoxidase (PAP)
Alkaline phosphatase - anti-Alkaline phosphatase (APAAP)

17. 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.

19. 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

20. 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

21. 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)

22. 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

23. Determining the Secretory Contents of the Neuroendocrine Neurones

24. 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)

26. GnRH and P-CREB

28. 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

29. THANKS