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Lecturer(s)
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Ovečka Miroslav, prof. Mgr. Ph.D.
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Course content
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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. Methods of plant sample preparation 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. 8. The fluorescent properties of conjugated fluorochromes, separation of excitation / emission spectra, multicolour colocalization, positive and negative controls. 9. Secondary antibodies, concentration, incubation time and conditions, blocking of unspecific binding, positive and negative controls. 10. Practical selection of primary and secondary antibodies, single-, double- and triple immunolabeling experiments, issues of antibody cross-reactivity and host specificity. 11. Preparation of cell monolayers, cell suspensions and unicellular organisms and wholemounts for immunofluorescence microscopy. 12. Tissue sectioning for immunofluorescence microscopy 13. Fluorescence properties of conjugated fluorophores, separation of excitation/emission spectra, multichannel imaging.
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Learning activities and teaching methods
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Lecture, Dialogic Lecture (Discussion, Dialog, Brainstorming), Demonstration
- Attendace
- 39 hours per semester
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Learning outcomes
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The course provides comprehensive theoretical and practical information to understand the significance of the Immunofluorescence methods in localization of different epitopes in prokaryotic and eukaryotic organisms and their practical application in the study of plant cells.
Students will gain theoretical basis for understanding the benefits of immunofluorescence in localization of different epitopes in situ with the ability to apply this knowledge in practice in plant cell biology.
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Prerequisites
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unspecified
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Assessment methods and criteria
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Written exam
Active participation on theoretical and practical parts, passing written test examination of the knowledge for the course credit with a score at least 60% and passing the final examination with a score at least 70% of points available.
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Recommended literature
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Bacallao R, Sohrab S, Phillips C. (2006). Guiding principles of specimen preservation for confocal fluorescence microscopy. In: Handbook of Biological Confocal Microscopy, Third Edition. Springer Science+Business Media, LLC, New York.
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Haseloff J, Dormand E-L, Brand AH. (1999). Live imaging of green fluorescent protein. Methods in Molecular Biology, vol. 122: Confocal Microscopy Methods and Protocols . Humana Press Inc., Totowa, NJ.
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Wymer CL, Beven AF, Boudonck K, Lloyd CW. (1999). Confocal microscopy of plant cells. Methods in Molecular Biology, vol. 122: Confocal Microscopy Methods and Protocols. Humana Press Inc., Totowa, NJ.
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