1. IMMUNOFLUORESCENCE Lab. 6

2. Immunofluorescence It is a technique that uses a fluorescent compound (fluorophore or fluorochrome) to indicate a specific antigen-antibody reaction If antibody molecules are tagged with a fluorescent dye and then binds to an antigen, this immune fluorescently labeled complex can be detected by colored light emission when excited by light of the appropriate wavelength Antibody molecules bound to antigens in cells or tissue sections can similarly be visualized 2

3. Applications of IF The presence of a specific antigen is determined by the appearance of localized color against a dark background This method is used for: Rapid identification of microorganisms in cell culture or infected tissue Antigens on neoplastic tissue & inside cells and CD antigens on T and B cells through the use of cell flow cytometry 3

4. Examples Anti-HBV preS2 (envelope proteins) Anti- AIF Ab Tubulin (green) M Mitochondria (red) Nonspecific stain for nuclei (blue) 4

5. Fluorophores Fluorophores are typically organic molecules with a ring structure They absorb light energy over a range of wavelengths that is characteristic for that compound This absorption of light causes an electron in the fluorescent compound to be raised to a higher energy level The excited electron quickly decays to its ground state, emitting the excess energy as a photon of light, which has a longer wavelength and lower energy This transition of energy is called fluorescence 5

6. Fluorescence  Ex Absorption Relaxation: Measured as the Fluorescence Lifetime (~ 1 – 25 ns) Em Fluorescence: Always at a higher wavelenth 6

7. Absorption & Emission Spectrums The range over which a fluorescent compound can be excited is termed its absorption spectrum (excitation) The range of emitted wavelengths for a particular compound is termed its emission spectrum The time interval between absorption of energy and emission of fluorescence is very short and can be measured in nanoseconds 7

8. Fluorphores Excitation & Emission Wavelenghts 8

9. Factors Affecting Fluorescence Increase fluorescence Structure Aromatic groups Rigidity Decrease fluorescence Temperature increase Heavy atoms in solvent Dissolved O 2

10. Types of Immunofluorescence Fluorescent staining can be categorized as direct or indirect, depending on whether the original antibody has a fluorescent tag attached Direct IF Indirect IF 10

11. Direct Immunofluorescence The antibody to the tissue antigen is conjugated with the fluorochrome and applied directly The antibody used is usually monoclonal antibody For example, to show the presence of virus antigens in tissue, fluorescence labeled antibodies are applied directly to the tissue When viewed with the fluorescence microscope, the tissue will be brightly stained 11

12. 12 Direct Immunofluorescence Ab to tissue Ag is labeled with fluorochrome Ag Fluorochrome Labeled Ab Tissue Section Ag

13. Indirect Immunofluorescence In this double-layer technique, the unlabeled antibody (primary Ab) is applied directly to the tissue and visualized by treatment with a fluorochrome- conjugated to anti-antibody (secondary antibody) The secondary antibody is anti-species antibody which is raised against the species where the primary antibody was produced It is a polyclonal antibody 13

14. 14 Indirect Immunofluorescence Ab to tissue Ag is unlabeled Fluorochrome-labeled anti-Ab is used to detect binding of the first Ab Fluorochrome Labeled Anti-Ab Tissue Section Unlabeled Ab Ag

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16. Direct & Indirect IF 16 DirectIndirect Fix specimen on slide Add labeled antibody specific for the desired antigen Look for fluorescence Fix specimen on slide Add primary antibody, specific for the desired antigen Add secondary labeled antibody Look for fluorescence

17. Advantages of Indirect IF The fluorescence is brighter than with the direct test since several fluorescent anti-immunoglobulins bind on to each of the antibody molecules present in the first layer Even when many sera have to be screened for specific antibodies it is only necessary to purchase a single labeled reagent The primary antibody does not need to be conjugated with a fluorochrome Because the supply of primary antibody is often a limiting factor, indirect methods avoid the loss of antibody that usually occurs during the conjugation reaction 17

18. Controls Reagent and tissue controls are necessary for the validation of immunofluorescence staining results Without their use, interpretation of staining would be haphazard and the results of doubtful value More specifically, controls determine if the staining protocols were: followed correctly whether day-to-day and worker-to-worker variations have occurred and that reagents remain in good working order 18

19. Controls In carrying out an immunofluorescence experiment one has to be confident that the reaction is specific and that the Ab is in fact binding selectively to the target Ag and not to other components of the cell or other closely related Ags In addition if no fluorescence is observed with the probe does this mean that: the Ag is not present or it mean that there may be a problem with preparation or with the tissue itself If the correct controls are included in the experiment we can, with high certainty, answer these questions 19

20. Positive and Negative Controls Negative Tissue Controls: Specimens serving as negative controls must be processed (fixed, embedded) identically to the unknown, but do not contain the target antigen If a signal is detected then this suggests that a problem exists within your technique or protocol Positive Tissue Controls: Again, these controls must be processed identically to the specimen but contain the target antigen If a signal is not detected then this suggests the problem exists within your technique, protocol or reagent 20

21. DETECTION OF SIGNAL 21

22. Fluorescence Instrument Instrument for detection of fluorescence consists of: Light source Xenon Arc Lamp or mercury vapor lamp Laser Wavelength selector Excitation filter Emission filter Detector Signal processor Emission filter 22 Xenon Arc Lamp

23. Fluorescence Microscope It is a microscope that uses fluorescence to generate an image The combination of exciter filter, dichroic mirror and emission filter should be selected according to the fluorochrome label The 3 components are usually built into a single module called the filter block 23

24. Fluorescence Microscope 24

25. Principles of confocal microscopy A focused laser beam serves as a high intensity light source Light reflected or fluorescence emitted by the specimen is allowed to pass through a pinhole that filters light coming from outside (above and below) of the focal plane A sensitive detector (photomultipler) behind a pinhole to measure the intensity of light The laser beam, the pinhole and detector scan through the specimen to build up an image on a monitor

26. Use of confocal microscope Performs optical sectioning of thick samples Three dimensional image reconstruction Detects very weak fluorescent signals Selective photobleaching

27. Quenching, Bleaching & Saturation Quenching is when excited molecules relax to ground states via nonradiative pathways avoiding fluorescence emission (vibration, collision, intersystem crossing) Molecular oxygen quenches by increasing the probability of intersystem crossing Photobleaching is defined as the irreversible destruction of an excited fluorophore

28. Fluorescence ENERGY S0S0 S1S1 S2S2 T2T2 T1T1 ABS FL I.C. ABS - AbsorbanceS 0.1.2 - Singlet Electronic Energy Levels FL - FluorescenceT 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal ConversionIsC - Intersystem CrossingPH - Phosphorescence IsC PH [Vibrational sublevels] Jablonski Diagram Vibrational energy levels Rotational energy levels Electronic energy levels Singlet StatesTriplet States fast slow (phosphorescence) Much longer wavelength (blue ex – red em) Triplet state

29. Indirect Fluorescence Assay For Mumps Virus IgG Antibody 29

30. Introduction and Summary of Test Procedures Mumps, an acute, contagious disease, is generally characterized clinically by parotitis Invasion of the central nervous system, testes, ovaries, and other visceral organs can accompany the infection The introduction of a mumps virus vaccine in 1967 has resulted in a decline in the incidence of mumps But because of the vaccine's restricted use, mumps will remain a common worldwide problem 30

31. Introduction and Summary of Test Procedures Laboratory confirmation of mumps infection is usually not required in those patients with characteristic parotitis However, the two most common complications, meningoencephalitis and orchitis, can occur without the classic parotitis In these cases, laboratory detection is necessary to confirm mumps infection 31

32. Principle of the Test Fluorescent antibody assays use the indirect method of antibody detection and titer determination Patient serum or plasma samples are applied to cultured cells containing inactivated viral antigens provided on wells on glass microscope slides During a 30 minute incubation, antibody specific for mumps virus antigens forms an antigen/antibody complex with the mumps virus antigens in the infected cells 32

33. Principle of the Test In a brief washing step, nonspecific antibody and other unreacted serum proteins are eliminated Fluorescein-conjugated goat antihuman IgG is then applied to the wells of the glass slide The anti-IgG conjugate combines with human IgG, if present, during a 30 minute incubation After a brief wash to remove unreacted conjugate, the slides are viewed by fluorescence microscopy A positive antibody reaction is denoted by bright green fluorescence at the antigen sites 33

34. Controls Mumps Virus IgG Positive Control: Each vial contains 0.5 ml mumps virus IgG antibody positive human control This component is a ready for use liquid at a 1:10 working dilution Mumps Virus IgG Negative Control: Each vial contains 0.5 ml mumps virus IgG antibody negative human control This component is a ready for use liquid at a 1:10 working dilution 34

35. Procedure 1. For qualitative IgG antibody determination, prepare a 1:10 screening dilution of each test sample in PBS Prepare all dilutions in a minimum volume of 0.10 ml with PBS as the diluent 2. For quantitative titration of sera, prepare two-fold serial dilutions of the serum sample in PBS, starting with a 1:10 dilution, and adding equal volumes of diluted serum or plasma and PBS for each consecutive dilution 3. Remove slides from protective pouch and apply 1 drop (approximately 20 µl) of the diluted test sample(s) to each well Add sufficient volume to completely cover each well, but cross-mixing of contents between wells should not occur Note: Each day’s test run requires one well each for positive control, negative control, and PBS (conjugate control) 35

36. Procedure 4. Incubate the slides in a moist chamber for 30 minutes at 37°C 5. Rinse the slides in a light stream of buffer Avoid directing the stream at the wells 6. Wash the slides for 10 minutes with a change of PBS solution after 5 minutes Agitate the slides by moving the rack up and down in the buffer 7. Blot the paint mask surrounding the test wells with the special blotters 36

37. Procedure 8. Apply one drop of the ready to use conjugate to each test well 9. Repeat steps 4 (incubation), 5 (PBS rinse), 6 (10 minute PBS wash), and 7 (blot) 10. Apply the glycerol mounting media and 22 X 50 mm glass coverslip 11. Observe the reactivity under fluorescence microscopy 37

38. Interpretation of Results Bright green fluorescent staining of the infected cells denotes a mumps virus IgG antibody positive reaction Absence of specific fluorescent staining of the infected cells denotes a mumps virus IgG antibody negative reaction Fluorescence found in both infected and uninfected cells, test sample is exhibiting a nonspecific reaction 38 Positive Negative

39. https://www.youtube.com/watch?v=23KXl3wPdP Y https://www.youtube.com/watch?v=23KXl3wPdP Y https://www.youtube.com/watch?v=g6Q1eel3YHs 39