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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. Basic terminology, concept of immunodetection, its use at the tissue and cellular level, principles and methods of epitope detection, immunofluorescence, immunohistochemistry, immunocytochemistry. 2. Basic concepts of the use of immunofluorescence at the tissue and cellular level, principles of epitope detection in cells in situ and in isolated fractions. Comparison with other methods of fluorescence detection. Principles and methods of detection of proteins and other molecules. 3. Basic methods of detection using direct and indirect immunofluorescence. Detection systems based on the principle of specific antibodies and the biotin-streptavidin system. Conjugation of labels, selectivity and specificity. Detection using fluorochromes and comparison with methods using conjugated enzymes, colloidal particles of gold and silver. 4. Practical use of fluorescence for antibody detection, principle of immunofluorescence labeling. Primary and secondary antibodies. Antibody concentration, incubation time and conditions, blocking of nonspecific binding, positive and negative controls. 5. Methods of sample preparation for immunofluorescence. Methods of fixation by physical, chemical and combined methods, ensuring the availability of epitopes for antibodies. Immunofluorescence microscopy of whole biological objects and cut samples. 6. Video sequences of complete immunofluorescence and immunohistochemical labeling procedures according to verified protocols. 7. Immunological basics of the use of antibodies in cell biology, structure, distribution, function and types of antibodies, selectivity, specificity, validation. 8. Secondary antibodies, specificity, conjugates. 9. Examples of practical selection of primary and secondary antibodies, experiments with single, double and triple immunolabeling, issues of antibody cross-reactivity and host specificity. 10. Protocols for immunofluorescence microscopy: I. Cell monolayers, suspensions, wholemounts. 11. Protocols for immunofluorescence microscopy: II. Mounting and sectioning of samples for immunofluorescence microscopy. 12. Fluorescence properties of conjugated fluorophores, separation of excitation/emission spectra, multichannel imaging of immunofluorescence preparations.
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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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Provides comprehensive theoretical and practical information to understand the significance of the methods and their practical application in the study of cells and biological samples.
Students will gain the theoretical foundations to understand the benefits of immunofluorescence in cell biology, histology, and developmental biology with the ability to apply this knowledge practically.
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Prerequisites
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unspecified
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Assessment methods and criteria
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Written exam
Participation, obtaining a credit with at least 70% of points from the written test, written exam with the requirement to obtain at least 60% of points.
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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.
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Hammers C., M., Stanley J., R.. (2014). Antibody Phage Display: Technique and Applications. Journal of Investigative Dermatology.
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Harms, P.V. et al. (2023). Multiplex Immunohistochemistry and Immunofluorescence: A Practical Update for Pathologists. Mod Pathol. 2023;36:100197. doi: 10.1016/j.modpat.2023.100197.
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Rüker, F., Wozniak-Knopp, G. (2020). Introduction to Antibody Engineering (Learning Materials in Biosciences).
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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.
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