Immunofluorescence is a technique allowing the visualization of a specific antigen by bindIng a specific antibody chemically conjugated with a fluorescent dye.

1. Immunofluorescence
Ranjeet Kumar Taram
Msc ll Sem (Microbiology )
S.o.S. in Life Science
Pt Ravishankar Shukla University Raipur

2. Introduction
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Immunofluorescence is a technique allowing
the visualization of a specific antigen by
bindIng a specific antibody chemically
conjugated with a fluorescent dye such as
fluorescin isothiosynate.
The most commonly used fluorescent dye are
fluorescin and rhodamin.
Other dye such as phycoerythrin and
phycobiliproteins, an intensely colored and
highly fluorescent pigment.
This molecules can be conjugated to the Fc
region of an antibody molecule without
affecting the specificity of the antibody and
visualize the immune complex.
FLUORESCENCE
MICROSCOPE
(SoS IN LIFE SCIENCES)

3. Principle
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Fluorescence is the property of the
certain molecules to absorb light at one
wavelength and emit light at larger
wavelength when it is illuminated by light
of a different wavelength.
the fluorescent can be visualized using
fluorescence microscopy. The
immunofluorescence technique allows
for the visualization of of the presence
as well as the distribution of target
molecules in a sample.
Each of fluorescent dye or
fluorochrome below absorbs light at one
wavelength and emits light at a larger
wavelength.

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Fluorescin, an organic dye that is most widely used labeled for
immunofluorescence procedure, absorbs blue light (490nm) and
emits an intense yellow green fluorescence (517nm)
Rhodamin another organic dye absorbs in the yellow green range
(515nm) and emits a deep red fluorescence (546nm)
Phycoerythrin :-It is an efficient absorber of light and a brilliant
emitter of red fluorescence.
In this technique antibody molecules are tagged with a fluorescent
dye.
When this antibody molecules bind with antigen form immune
complex and this immune complexes are easily visualized or
detected by the color light emission when excited by the light of the
appropriate wavelength.

5. Sample preparation for immunofluorescence

6. Types of
immunofluorescence
Direct immunofluorescence - In
this method, only a single antibody
is used that is chemically linked to
a fluorochrome. The antibody
recognizes the target molecules
and binds to it, and form immune
complex that is detected via
microscopy.

7. Advantages of direct
immunofluorescence
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direct conjugation of the antibody
reduce the number of steps in the staining procedure
making the process faster and reduce background signal
by avoiding some is used with antibody cross reactivity
direct visualisation

8. indirect immunofluorescence
indirect immunofluorescence
uses two antibodies the
unlabelled primary antibody
specifically binds to the target
molecule and the secondary
antibody which carries the
flow Chrome recognise the
primary antibody and bind to it.

9. Advantages of indirect
immunofluorescence
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indirect methods avoid the loss of antibody that is usually
occurs during the conjugation
indirect methods increase the sensitivity of staiming
because multiple molecules of the fluorochrome reagent
bind to each primary antibody molecule.
Requires only one Level antibody to identify many proteins
sim labelled secondary antibody can be used to Wine bind
too many different proteins

10. Applications
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to identify the number of sub population of lymphocytes
notably the cd4 and cd8 T-cell population.
Identifying bacterial species
detecting antigen antibody complex in autoimmune
disease
detecting complement component in tissues
To localising hormones and other cellular product stain in
situ study of evolution of cells in suspension cultured cell,
tissue beads.

11. Limitations
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Photobleaching: The cells lose their emission property i.e intensity of
fluorescence with time.
increased background noises: Due to multiple attachment of
Secondary antibody.
Non specific fluorescence: Involves the loss of a Probe's specifity
due to flurophore , from improper fixation, or from dried out specimen.

12. Reference
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Goldsby, Richard A., Thomas J. Kindt, and Barbara A. Osborne. "Kuby
immunology. 4th." USA. W (2000).
Im, Kyuseok, et al. "An Introduction to Performing Immunofluorescence
Staining." Biobanking. Humana Press, New York, NY, 2019 299-311
Wilson, K., & Walker, J. (Eds.). (2000). Principles and techniques of
practical biochemistry. Cambridge University Press.

13. Thank you