Immunofluorescence

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Photomicrograph of a histological section of human

skin prepared for direct immunofluorescence using an

anti-IgA antibody. The skin is from a patient with
Henoch–Schönlein purpura: IgA deposits are found in
the walls of small superficial capillaries (yellow
arrows). The pale wavy green area on top is the
epidermis the bottom fibrous area is the dermis
epidermis, the bottom fibrous area is the dermis.

These figures demonstrates the basic mechanism of

immunofluorescence. Primary immunofluorescence is
depicted on the left, which shows an antibody with a
fluorophore group bound to it directly binding to the
epitope of the antigen for which it is specific. Once the
antibody binds to the epitope, the sample can be
viewed under fluorescent microscope to confirm the
presence of the antigen in the sample. Conversely,
secondary immunofluorescence is depicted to the
right, which shows that first an untagged primary
antibody binds to the epitope of the antigen in a

mechanism similar to the one described above.

However, after the primary antibodies have bound to
their target, a secondary antibody (tagged with a
fluorophore) comes along. This secondary antibody’s
binding sites are specific for the primary antibody
that’s already bound to the antigen, and therefore the
secondary antibody binds to the primary antibody. This
method allows for more fluorophore-tagged antibodies
to attach to their target, thus increasing the fluorescent
signal during microscopy.

Immunofluorescence is a technique used

for light microscopy with a fluorescence
microscope and is used primarily on
microbiological samples. This technique
uses the specificity of antibodies to their
antigen to target fluorescent dyes to
specific biomolecule targets within a cell,
and therefore allows visualization of the
distribution of the target molecule through
the sample. The specific region an
antibody recognizes on an antigen is
called an epitope.[1] There have been
efforts in epitope mapping since many
antibodies can bind the same epitope and
levels of binding between antibodies that
recognize the same epitope can vary.[2]
Additionally, the binding of the fluorophore
to the antibody itself cannot interfere with
the immunological specificity of the
antibody or the binding capacity of its
antigen.[3] Immunofluorescence is a widely
used example of immunostaining (using
antibodies to stain proteins) and is a
specific example of
immunohistochemistry (the use of the
antibody-antigen relationship in tissues).
This technique primarily makes use of
fluorophores to visualise the location of
the antibodies.[4]

Immunofluorescence can be used on

tissue sections, cultured cell lines, or
individual cells, and may be used to
analyze the distribution of proteins,
glycans, and small biological and non-
biological molecules. This technique can
even be used to visualize structures such
as intermediate-sized filaments.[5] If the
topology of a cell membrane has yet to be
determined, epitope insertion into proteins
can be used in conjunction with
immunofluorescence to determine
structures.[6] Immunofluorescence can
also be used as a "semi-quantitative"
method to gain insight into the levels and
localization patterns of DNA methylation
since it is a more time consuming method
than true quantitative methods and there
is some subjectivity in the analysis of the
levels of methylation.[7]
Immunofluorescence can be used in
combination with other, non-antibody
methods of fluorescent staining, for
example, use of DAPI to label DNA. Several
microscope designs can be used for
analysis of immunofluorescence samples;
the simplest is the epifluorescence
microscope, and the confocal microscope
is also widely used. Various super-
resolution microscope designs that are
capable of much higher resolution can
also be used.[8]

Types

Photomicrograph of a histological section of human

skin prepared for direct immunofluorescence using an
anti-IgG antibody. The skin is from a patient with
systemic lupus erythematosus and shows IgG deposit
at two different places: The first is a band-like deposit
along the epidermal basement membrane ("lupus band
test" is positive). The second is within the nuclei of the
epidermal cells (anti-nuclear antibodies).
Preparation of fluorescence

To make fluorochrome-labeled antibodies,

a fluorochrome must be conjugated
("tagged") to the antibody. Likewise, an
antigen can also be conjugated to the
antibody with a fluorescent probe in a
technique called fluorescent antigen
technique. Staining procedures can apply
to both fixed antigen in the cytoplasm or to
cell surface antigens on living cells, called
"membrane immunofluorescence". It is
also possible to label the complement of
the antibody-antigen complex with a
fluorescent probe. In addition to the
element to which fluorescence probes are
attached, there are two general classes of
immunofluorescence techniques: primary
and secondary. The following descriptions
will focus primarily on these classes in
terms of conjugated antibodies.[3]

There are two classes of

immunofluorescence techniques, primary
(or direct) and secondary (or indirect).

Primary (direct)

Primary (direct) immunofluorescence uses

a single, primary antibody, chemically
linked to a fluorophore. The primary
antibody recognizes the target molecule
(antigen) and binds to a specific region
called the epitope. The attached
fluorophore can be detected via
fluorescent microscopy, which, depending
on the messenger used, will emit a
specific wavelength of light once
excited.[9] Direct immunofluorescence,
although somewhat less common, has
notable advantages over the secondary
(indirect) procedure. The direct
attachment of the messenger to the
antibody reduces the number of steps in
the procedure, saving time and reducing
non-specific background signal.[10] This
also limits the possibility of antibody
cross-reactivity and possible mistakes
throughout the process.

However, some disadvantages do exist in

this method. Since the number of
fluorescent molecules that can be bound
to the primary antibody is limited, direct
immunofluorescence is substantially less
sensitive than indirect
immunofluorescence and may result in
false negatives. Direct
immunofluorescence also requires the use
of much more primary antibody, which is
extremely expensive, sometimes running
up to $400.00/mL.
Secondary (indirect)

A fluorescent stain for actin in the smooth muscle of

the skin.

Secondary (indirect) immunofluorescence

uses two antibodies; the unlabeled first
(primary) antibody specifically binds the
target molecule, and the secondary
antibody, which carries the fluorophore,
recognizes the primary antibody and binds
to it. Multiple secondary antibodies can
bind a single primary antibody. This
provides signal amplification by increasing
the number of fluorophore molecules per
antigen.[10] This protocol is more complex
and time-consuming than the primary (or
direct) protocol above, but allows more
flexibility because a variety of different
secondary antibodies and detection
techniques can be used for a given
primary antibody.[10]

This protocol is possible because an

antibody consists of two parts, a variable
region (which recognizes the antigen) and
constant region (which makes up the
structure of the antibody molecule). It is
important to realize that this division is
artificial and in reality the antibody
molecule is four polypeptide chains: two
heavy chains and two light chains. A
researcher can generate several primary
antibodies that recognize various antigens
(have different variable regions), but all
share the same constant region. All these
antibodies may therefore be recognized by
a single secondary antibody. This saves
the cost of modifying the primary
antibodies to directly carry a fluorophore.

Different primary antibodies with different

constant regions are typically generated by
raising the antibody in different species.
For example, a researcher might create
primary antibodies in a goat that recognize
several antigens, and then employ dye-
coupled rabbit secondary antibodies that
recognize the goat antibody constant
region ("rabbit anti-goat" antibodies). The
researcher may then create a second set
of primary antibodies in a mouse that
could be recognized by a separate "donkey
anti-mouse" secondary antibody. This
allows re-use of the difficult-to-make dye-
coupled antibodies in multiple
experiments.

Limitations
As with most fluorescence techniques, a
significant problem with
immunofluorescence is photobleaching.
Loss of activity caused by photobleaching
can be controlled by reducing or limiting
the intensity or time-span of light
exposure, by increasing the concentration
of fluorophores, or by employing more
robust fluorophores that are less prone to
bleaching (e.g., Alexa Fluors, Seta Fluors,
or DyLight Fluors). Some problems that
may arise from this technique include
autofluorescence, extraneous undesired
specific fluorescence, and nonspecific
fluorescence. Autofluorescence includes
fluorescence emitted from the sample
tissue or cell itself. Extraneous undesired
specific fluorescence occurs when a
targeted antigen is impure and contains
antigenic contaminants. Nonspecific
fluorescence involves the loss of a probe's
specificity due to fluorophore, from
improper fixation, or from a dried out
specimen.[3]

Immunofluorescence is only limited to

fixed (i.e., dead) cells when structures
within the cell are to be visualized because
antibodies do not penetrate the cell
membrane when reacting with fluorescent
labels. Antigenic material must be fixed
firmly on the site of its natural localization
inside the cell.[3] Intact antibodies can also
be too large to dye cancer cells in vivo.[11]
Their size results in slow tumor
penetration and long circulating half-life.
Research has been done investigating the
use of diabodies to get around this
limitation.[11] Proteins in the supernatant
or on the outside of the cell membrane
can be bound by the antibodies; this
allows for living cells to be stained.
Depending on the fixative that is being
used, proteins of interest might become
cross-linked and this could result in either
false positive or false negative signals due
to non-specific binding.
An alternative approach is using
recombinant proteins containing
fluorescent protein domains, e.g., green
fluorescent protein (GFP). Use of such
"tagged" proteins allows determination of
their localization in live cells. Even though
this seems to be an elegant alternative to
immunofluorescence, the cells have to be
transfected or transduced with the GFP-
tag, and as a consequence they become at
least S1 or above organisms that require
stricter security standards in a laboratory.
This technique involves altering the
genetic information of cells.[12]

Advances
Many improvements to this method lie in
the improvement of fluorescent
microscopes and fluorophores. Super-
resolution methods generally refer to a
microscope's ability to produce resolution
below the Abbe limit (a limit placed on
light due to its wavelength). This
diffraction limit is about 200-300 nm in the
lateral direction and 500-700 nm in the
axial direction. This limit is comparable or
larger than some structures in the cell, and
consequently, this limit prevented
scientists from determining details in their
structure.[13] Super-resolution in
fluorescence, more specifically, refers to
the ability of a microscope to prevent the
simultaneous fluorescence of adjacent
spectrally identical fluorophores.[14] This
process effectively sharpens the point-
spread function of the microscope.[13]
Examples of recently developed super-
resolution fluorescent microscope
methods include stimulated emission
depletion (STED) microscopy, saturated
structured-illumination microscopy (SSIM),
fluorescence photoactivation localization
microscopy (FPALM), and stochastic
optical reconstruction microscopy
(STORM).[15]

See also
 Cutaneous conditions with
immunofluorescence findings
Immunochemistry
Patching and Capping

References
1. Mandrell, R. E.; Griffiss, J. M.; Macher, B.
A. (1988-07-01). "Lipooligosaccharides
(LOS) of Neisseria gonorrhoeae and
Neisseria meningitidis have components
that are immunochemically similar to
precursors of human blood group antigens.
Carbohydrate sequence specificity of the
mouse monoclonal antibodies that
recognize crossreacting antigens on LOS
and human erythrocytes" . Journal of
Experimental Medicine. 168 (1): 107–126.
doi:10.1084/jem.168.1.107 . ISSN 0022-
1007 . PMC 2188965 . PMID 2456365 .
2. Ladner, Robert C. (2007-01-01). "Mapping
the Epitopes of Antibodies". Biotechnology
and Genetic Engineering Reviews. 24 (1):
1–30. CiteSeerX 10.1.1.536.6172 .
doi:10.1080/02648725.2007.10648092 .
ISSN 0264-8725 .
3. Akiyoshi., Kawamura (1983-01-01).
Immunofluorescence in medical science :
with 28 tab. Springer u.a. ISBN 978-
3540124832. OCLC 643714056 .
4. "Immunofluorescence" . Protocol Online.
5. Franke, W. W.; Schmid, E.; Osborn, M.;
Weber, K. (1978-10-01). "Different
intermediate-sized filaments distinguished
by immunofluorescence microscopy" .
Proceedings of the National Academy of
Sciences of the United States of America.
75 (10): 5034–5038.
doi:10.1073/pnas.75.10.5034 . ISSN 0027-
8424 . PMC 336257 . PMID 368806 .
6. Wang, Honggang; Lee, Eun-Woo; Cai,
Xiaokun; Ni, Zhanglin; Zhou, Lin; Mao,
Qingcheng (2008-12-30). "Membrane
Topology of the Human Breast Cancer
Resistance Protein (BCRP/ABCG2)
Determined by Epitope Insertion and
Immunofluorescence" . Biochemistry. 47
(52): 13778–13787.
doi:10.1021/bi801644v . ISSN 0006-2960 .
PMC 2649121 . PMID 19063604 .
7. Çelik, Selcen (2015-01-01).
"Understanding the complexity of antigen
retrieval of DNA methylation for
immunofluorescence-based measurement
and an approach to challenge" . Journal of
Immunological Methods. 416: 1–16.
doi:10.1016/j.jim.2014.11.011 .
PMID 25435341 .
8. "Immunofluorescence Method" .
Davidson College.
9. "Immunohistochemical Staining
Methods" (PDF). IHC Guidebook (Sixth ed.).
Dako Denmark A/S, An Agilent
Technologies Company. 2013.
10. Fritschy J, Härtig W (2001).
Immunofluorescence . eLS.
doi:10.1038/npg.els.0001174 . ISBN 978-0-
470-01590-2.
11. Sonn GA, Behesnilian AS, Jiang ZK,
Zettlitz KA, Lepin EJ, Bentolila LA, Knowles
SM, Lawrence D, Wu AM, Reiter RE (2016).
"Fluorescent Image-Guided Surgery with an
Anti-Prostate Stem Cell Antigen (PSCA)
Diabody Enables Targeted Resection of
Mouse Prostate Cancer Xenografts in Real
Time" . Clinical Cancer Research. 22 (6):
1403–12. doi:10.1158/1078-0432.CCR-15-
0503 . PMC 4794340 . PMID 26490315 .
12. Chalfie, Martin (1995-10-01). "Green
Fluorescent Protein". Photochemistry and
Photobiology. 62 (4): 651–656.
doi:10.1111/j.1751-1097.1995.tb08712.x .
ISSN 1751-1097 .
13. Huang, Bo; Bates, Mark; Zhuang,
Xiaowei (2009-06-02). "Super-Resolution
Fluorescence Microscopy" . Annual Review
of Biochemistry. 78: 993–1016.
doi:10.1146/annurev.biochem.77.061906.0
92014 . PMC 2835776 . PMID 19489737 .
14. 1959-, Diaspro, Alberto; van, Zandvoort,
Marc A. M. J. (2016-11-03). Super-
resolution imaging in biomedicine.
ISBN 9781482244359. OCLC 960719686 .
15. Leung, Bonnie O.; Chou, Keng C. (2011-
09-01). "Review of Super-Resolution
Fluorescence Microscopy for Biology".
Applied Spectroscopy. 65 (9): 967–980.
doi:10.1366/11-06398 . ISSN 0003-7028 .
PMID 21929850 .

External links
Images associated with autoimmune
diseases at University of Birmingham
Immunofluorescence Staining Protocol
Overview at Davidson College
Immunofluorescence at the US
National Library of Medicine Medical
Subject Headings (MeSH)
 SynD - Automatic synapse and neurite
detection in immuno-fluorescence
images

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