Modern Immunofluorescence Techniques

Day 1

1. History of the development and introduction of immunofluorescence in biological research, theoretical base and the main advantages of immunofluorescence. Comparison with other methods of fluorescence detection.

2. Basic concept of the use of immunofluorescence at the tissue and cellular levels, the principles of epitopes detection in cells and in isolated fractions. Principles and methods for detecting proteins and other molecules.

3. Basic methods of detection by direct and indirect immunofluorescence. Detection systems based on specific antibodies and biotin-streptavidin system. Conjugation of markers, selectivity and specificity. Detection using fluorochromes and comparison with the methods of conjugated enzymes, colloidal gold and silver particles.

4. Methods of sample preparation for immunofluorescence. Methods of fixation by physical, chemical and combined methods, the availability of epitopes for antibodies. Immunofluorescence microscopy of whole plant organs and sectioned samples.

5. Practical preparation of plant samples for the immunofluorescence detection, fixation, digestion of the cell wall and plasma membrane permeabilization.

6. Immunological principles of antibodies use in cell biology, structure, classification, function and types of antibodies.

7. Uses of antibodies in immunofluorescence microscopy. Primary and secondary antibodies. The antibody concentration, incubation times and conditions, blocking of non-specific binding. The fluorescent properties of conjugated fluorochromes, separation of excitation / emission spectra, multicolour colocalization, positive and negative controls.

8. Various methods of counterstaining of cellular structures in plant samples after immunofluorescent labelling for microscopic identification. Methods for the preparation of stable microscopic slides by closing the sample in a suitable medium, preventing bleaching and fading of fluorescence during microscopic imaging.

9. Practical immunostaining of prepared fixed plant samples using a predetermined primary and secondary antibodies, the final preparation of specimens for microscopic observation.

10. Imaging of prepared objects in the fluorescence microscope and confocal laser scanning microscope. Demonstration of the specificity, stability and intensity of fluorescence labelling.

11. Confocal microscopy with a spinning disk, evaluation of changes in structure and mobility of cytoskeleton and endosomes

12. Assessment of methodological approaches and data obtained in the presentation of interactive protocols.

Some terms

• Fluorescence

• Phosphorescence Luminescence

Definition: Interactions between light and matter that result to the secondary emission of light. The secondary light (emission light) has wavelength (em) smaller that the wavelength used to evoke it (excitation light; exc). Substances that interact with light in this manner are complex aromatic compounds with extended molecular orbitals.

Understanding the π-bond and molecular orbitals

Orbital: Solutions of the wave function of single electrons; defines the space of probability that an electron can be found (Heisenberg Uncertainty Principle) π-bond: constructive interference of two or more p-orbitals. Very common in benzene ring based chemicals Molecular orbital: Delocalized electron cloud in periodically unsaturated chemical structures

Fluorescein

Fluorophore

Benzene ring

http://www.ibiology.org/ibioeducation/taking-courses/ibiology-microscopy-short-course/fluorescent-probes-2.html

Fluorophores: Chemical

π-electron ground state (LUMO) S0

S1

S2

hν1

Ene

rgy

Energy loss with no photophysical interest

HOMO hν2

Π-electrons are fairly unstable and excitable if fed with high energy

Some terms

Some terms

• Jablonski diagram

• Stokes shift

Stokes shift is the difference between λem – λexc and is characteristic for each fluorophore. Big Stokes shifts are associated with high energy loss during emission

Some terms

• Quantum efficiency

Quantum efficiency is characteristic for every fluorophore and tells us how bright the fluorophore will be. However brightness is also associated with the spectral characteristics of the photon emitted

Landmarks

• Fluorescence

• Phosphorescence

Luminescence

Matlaline was the first fluorophore to be discovered. It was isolated from the wood of Eysenhartdia polystachya in 1560.

Landmarks

• 1819: Fluorescence in fluorites (minerals containing fluorine)

• 1833: Fluorescence of chlorophyll

• 1845: Fluorescence of quinine

• 1871: Synthesis of fluorescein

Landmarks

• First fluorimetric application was the quantitative analysis of Al complexed to morin

• Tracing ground water with fluorescein

Landmarks

• 1901-1904: Development of first fluorescence microscopes (Otto Heimstaedt and Heinrich Lehmann)

• 1914: Dye binding of living cells (Stanislav Von Prowazek)

Landmarks

• 1941: Birth of Immunofluorescence. Coupling of fluorescein to antibodies by Albert Coons

• 1962: Osamu Shimomura discovered Green Fluorescent Protein

Antibodies

• Antibodies are transmembrane or secreted proteins of the immune system produced by vertebrates

Antibodies

• Anti-idiotypic (Idiotypes are antibodies that recognize different specific epitopes. The thing that determines the idiotype is way at the end of the variable region)

• Anti-isotypic (Antibody isotypes are the same thing as antibody classes. There are 5 major isotypes: IgM, IgD, IgG, IgE, and IgA. The difference between these isotypes lies in the heavy chain (Mu, Delta, Gamma, Epsilon, or Alpha).

• Anti-allotypic (Allotypes represent the genetically determined differences in antibodies between species)

Antibodies Structure nomenclature

ANTIGEN BINDING

Typical IgG molecule: C areas are invariable between different B-cells. V areas are cell specific

Antibodies

Structure nomenclature

1. Heavy chain

2. Light chain

3. Heavy chain variable (constant) region

4. Light chain variable (constant) region

5. Fab fragment (by protease treatment)

6. Fc fragment (by protease treatment)

Antibodies

• Immunoglobulins can be transmembrane (T-cells) or secreted (B-cells)

• Soluble immunoglobulins can be isolated from sera of immunized animals, from ascites fluids, or from culture supernates

• Such soluble immunoglobulins (and most commonly IgGs) are used in immunochemical applications

ANTIBODIES

SPECIFICITY

• The type of the antigen may vary: – Protein/peptide

– Lipooligosaccharide

– DNA (e.g., lupus erythematosus)

– Small molecules • For the latter, immune response is evoked only if

attached to bigger molecules (e.g., keyhole limpet hemocyanin, some albumin etc).

• The small molecule is called hapten and the big one is called hapten carrier

Antibodies

CLONALITY

Short history

• 1900: Antibody formation theory (Paul Ehrlich)

• 1938: Antibody-antigen interaction (John Marrack)

• 1948: Discovery of soluble immunoglobulins produced by B-cells

• 1975: Production of monoclonal antibodies by Georges Koehler and Cesar Milstein

Antibodies

POLYCLONAL

Actually the term polyclonal antibody is technically wrong. Correctly we should say polyclonal mixture of immunoglobulins or mixture of different monoclonal immunoglobulins Rabbits (or other animals) are injected with the desired antigen mixed with Freund’s adjuvant. The latter is a stimulant of the immune response.

Freund's adjuvant is a solution of antigen emulsified in mineral oil and used as an immunopotentiator (booster). The complete form, Freund's Complete Adjuvant,(CFA or FCA) is composed of inactivated and dried mycobacteria (usually M. tuberculosis), whereas the incomplete form (IFA or FIA) lacks the mycobacterial components (hence just the water in oil emulsion). It is named after Jules T. Freund.

Antibodies

POLYCLONAL

• The end product is a mixture of immunoglobulins (preferably IgGs) with different specificity against the same antigen (please remember difference between antigen and epitope or antigenic determinant) IDIOTYPES

Antibodies

POLYCLONAL

Advantages

• High affinity

• Ease and speed of production

• Cheap

Disadvantage

• Specificity issues, dirty result

Antibodies

MONOCLONAL

1. Immunization of mice 2. Isolation of B-cells 3. Fusion of B-cells with myeloma cells

1. This may lead to formation of: B-cell – B-cell (produce IgGs but die) myeloma – myeloma (are immortal but do not produce IgGs) B-cell – myeloma (hybridoma) (they are both)

4. Selection of appropriate line 5. Scaling up production a. Establishment of cell cultures b. Ascites fluid

Antibodies

MONOCLONAL

Advantages

• Very high specificity (practically monospecific)

Disadvantages

• Cost

• Affinity

• Range of applications

Antibodies

Conjugates

• Chromogenic

• Fluorigenic

Chromogenic detection via antibodies requires their conjugation with appropriate enzymes • Peroxidase (oxidation of substrates – e.g., diaminobenzidine,

luminol – in the presence of H2O2) • Alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate

(BCIP) and nitroblue tetrazolium (NBT))

Antibodies

Examples of chromogenic HRP substrates

Antibodies

BCIP

NBT

Oxidative cleavage of NBT in a blue formazan precipitate in the presence of BCIP as oxidant

Antibodies

Conjugates

• Chromogenic

• Fluorigenic

Fluorigenic detection via antibodies requires their conjugation with appropriate fluorophores • Fluorescein, rhodamine etc

Immunofluorescence

Work Flow

1. Fix cells

2. Permeabilize cells

3. Incubate with conjugated antibodies

4. Go to the microscope

Immunofluorescence

• Direct immunofluorescence

• Indirect immunofluorescence

Immunofluorescence

• Direct immunofluorescence

• Indirect immunofluorescence

So called primary antibody is directly conjugated with fluorophore

Amplification: 1 antigenic site might be detected with 3-4 fluorophore molecules

Immunofluorescence

• Direct immunofluorescence

• Indirect immunofluorescence

So called primary antibody is detected via secondary antibody coupled to fluorophores • Principally primary antibody is monoclonal • Secondary antibody is polyclonal

Amplification: 1 antigenic site might be detected with 9-30 fluorophore molecules. Extreme amplification of signal, improved SNR

Immunofluorescence

Conjugates

Immunofluorescence

Some examples

With immunofluorescence labeling we can practically image indefinite number of epitopes in the same cell. Limitations are: Spectral overlap of different fluorophores and antibody allotypes that may be used

Immunofluorescence

Comparison with other detection methods

1. Affinity labeling

2. Fluorescent protein labeling

3. Chromogenic labeling

Immunofluorescence

• Affinity labeling

It relies on modifications of small molecules with fluorophores

1. Highly specific

2. High affinity

3. Poor labeling

Immunofluorescence

Phalloidin

Phalloidin is a fungal toxin that binds to and stabilizes actin filaments. Once conjugated with fluorophore can be used to stain AFs in animal, plant and fungal cells.

Immunofluorescence

• Due to signal amplification, immunofluorescent localization of AFs is sharper but not as specific

Immunofluorescence

• Fluorescent protein tagging

Immunofluorescence

• Time lapsed – dynamic – imaging and quantitation

• Very specific tagging

• Copious work for cloning and transformation

• Difficult for colocalization studies

Immunofluorescence

• Western Blot • ELISA • FACS