1. Immunofluorescence
Lab. 6

2. Immunofluorescence UV
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
phycoerythrin an intensely colored and highly fluorescent pigment obtained from algae, an efficient absorber of light (~30-fold greater than fluorescein), and a brilliant emitter of red fluorescence, stimulating its wide use as a label for immunofluorescence. UV
Antibody molecules bound to antigens in cells or tissue sections can similarly be visualized
The presence of a specific antigen is determined by the appearance of localized color against a dark background

3. Applications of IF 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
Detection of different proteins inside cells

4. Examples Tubulin (green) M Mitochondria (red) Nonspecific stain for
nuclei (blue)
Anti-HBV PreS2
(envelope proteins)
The HBV surface protein antigens (HBsAg) are comprised of three carboxyl co terminal HBs proteins termed large (LHBs), middle (MHBs) and small (SHBs, also called major) protein. LHBs and MHBs also share the highly hydrophobic, repetitive, membrane spanning S domain. In addition, MHBs has a 55 amino acid region called preS2.
Cells infected with adenovirus
infected cells in green and
nuclei stained with DAPI in blue.

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

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

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

8. Factors Affecting Fluorescence
Increase fluorescence
Structure
Aromatic groups
Rigidity
rigid structures have lower probability of collisions
Decrease fluorescence
Temperature increase
Heavy atoms in solvent ( Intersystem crossing)
Dissolved O2 ( Intersystem crossing)

9. Factors Affecting Fluorescence

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

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 Em
Ab to tissue Ag is labeled with fluorochrome Em Em Ex
Fluorochrome
Labeled Ab Y Ag Ag Ag
Tissue Section

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

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

16. 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. 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. 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. 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. Detection of signal

21. 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
Xenon Arc Lamp
Emission filter

22. 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
A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors.

23. Quenching & Bleaching
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.
Following absorption, molecules can relax via a non-radiative transition to the T1 rather than the S1 state - this is called an intersystem crossing

24. Indirect Fluorescence Assay For Mumps Virus IgG Antibody

25. 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.
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
Parotitis: التهاب الغدة النكفية

26. 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

27. 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

28. 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

29. 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
Positive
Negative