CRH/MIT Modern Techniques of Immunofluorescence

Lecturers: Miroslav Ovečka, Georgios Komis
Seminar: 3 hours/week
Credits: 4
Winter semester
Form of course completion: course credit, exam

• 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.
• 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.
• 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.
• 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.
• Practical preparation of plant samples for the immunofluorescence detection, fixation, digestion of the cell wall and plasma membrane permeabilization.
• Immunological principles of antibodies use in cell biology, structure, classification, function and types of antibodies.
• 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.
• 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.
• Practical immunostaining of prepared fixed plant samples using a predetermined primary and secondary antibodies, the final preparation of specimens for microscopic observation.
• Imaging of prepared objects in the fluorescence microscope and confocal laser scanning microscope. Demonstration of the specificity, stability and intensity of fluorescence labelling.
• Confocal microscopy with a spinning disk, evaluation of changes in structure and mobility of cytoskeleton and endosomes
• Assessment of methodological approaches and data obtained in the presentation of interactive protocols.