Immunohistochemistry Aulanni’am University of Brawijaya

Labelling_Conjugated

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

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.

Monoclonal & Polyclonal PolyclonalMonoclonal multi antigenic sitesOne 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 Reproducibility and standardization difficult Highly reproducible

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 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 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 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

Radioisotope labels Advantages  Flexibility  Sensitivity  Size Disadvantages  Toxicity  Shelf life  Disposal costs

Enzyme labels Advantages  Diversity  Amplification  Versatility Disadvantages  Lability  Size  Heterogeneity

Fluorescent labels Advantages  Size  Specificity  Sensitivity Disadvantages  Hardware  Limited selection  Background

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  Size  Sensitivity  S/N Disadvantages  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 Microtiter well EEEEE Specimen 2nd antibody E Substrate SP

ELISA (variation 1) Microtiter well Specimen Labeled antigen E EEE SP

ELISA (variation 2) Microtiter well Specimen Labeled antibody E EEEE E E E

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)

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

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

Double labelling

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)

INDIRECT IMMUNOFLUORESCENCE Some examples

Indirect Fluorescent Antibody Test 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.

Indirect Fluorescent Antibody Test 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

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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