Immunofluorescence

Immunofluorescence is a technique used

for light microscopy with a fluorescence
microscope and is used primarily on
biological 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]

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.

Diagram of primary and secondary

immunofluorescence: Primary
immunofluorescence involves an
antibody with a fluorophore emitting a
signal once bound to a specific
antigen's epitope. Secondary
immunofluorescence has a secondary
antibody with a fluorophore binding a
primary antibody specific to the
antigen's epitope.
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. This is accomplished
by a process which manipulates the
immune response of organism with
adaptive immunity. 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.

Main antinuclear antibody patterns on

immunofluorescence.[11]

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.[12]
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.[12] 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.[13]

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.[14] Super-resolution in
fluorescence, more specifically, refers to
the ability of a microscope to prevent the
simultaneous fluorescence of adjacent
spectrally identical fluorophores.[15] This
process effectively sharpens the point-
spread function of the microscope.[14]
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).[16]

Notable people
Cornelia Mitchell Downs (1892–1987),
microbiologist and journalist

See also
Cutaneous conditions with
immunofluorescence findings
Immunochemistry
Patching and Capping

References
1. Mandrell RE, Griffiss JM, Macher BA (July
1988). "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" (https://www.ncbi.nlm.nih.go
v/pmc/articles/PMC2188965) . The
Journal of Experimental Medicine. 168 (1):
107–126. doi:10.1084/jem.168.1.107 (http
s://doi.org/10.1084%2Fjem.168.1.107) .
PMC 2188965 (https://www.ncbi.nlm.nih.g
ov/pmc/articles/PMC2188965) .
PMID 2456365 (https://pubmed.ncbi.nlm.ni
h.gov/2456365) .
2. Ladner RC (2007-01-01). "Mapping the
epitopes of antibodies". Biotechnology &
Genetic Engineering Reviews. 24 (1): 1–30.
CiteSeerX 10.1.1.536.6172 (https://citeseer
x.ist.psu.edu/viewdoc/summary?doi=10.1.
1.536.6172) .
doi:10.1080/02648725.2007.10648092 (htt
ps://doi.org/10.1080%2F02648725.2007.10
648092) . PMID 18059626 (https://pubme
d.ncbi.nlm.nih.gov/18059626) .
S2CID 34595289 (https://api.semanticscho
lar.org/CorpusID:34595289) .
3. Akiyoshi K (1983-01-01).
Immunofluorescence in medical science :
with 28 tab. Springer u.a. ISBN 978-
3540124832. OCLC 643714056 (https://ww
w.worldcat.org/oclc/643714056) .
4. "Immunofluorescence" (http://www.protoco
l-online.org/prot/Immunology/Immunofluor
escence/) . Protocol Online.
5. Franke WW, Schmid E, Osborn M, Weber K
(October 1978). "Different intermediate-
sized filaments distinguished by
immunofluorescence microscopy" (https://
www.ncbi.nlm.nih.gov/pmc/articles/PMC3
36257) . Proceedings of the National
Academy of Sciences of the United States
of America. 75 (10): 5034–5038.
Bibcode:1978PNAS...75.5034F (https://ui.a
dsabs.harvard.edu/abs/1978PNAS...75.503
4F) . doi:10.1073/pnas.75.10.5034 (https://
doi.org/10.1073%2Fpnas.75.10.5034) .
PMC 336257 (https://www.ncbi.nlm.nih.go
v/pmc/articles/PMC336257) .
PMID 368806 (https://pubmed.ncbi.nlm.ni
h.gov/368806) .
6. Wang H, Lee EW, Cai X, Ni Z, Zhou L, Mao Q
(December 2008). "Membrane topology of
the human breast cancer resistance protein
(BCRP/ABCG2) determined by epitope
insertion and immunofluorescence" (http
s://www.ncbi.nlm.nih.gov/pmc/articles/PM
C2649121) . Biochemistry. 47 (52): 13778–
13787. doi:10.1021/bi801644v (https://doi.
org/10.1021%2Fbi801644v) .
PMC 2649121 (https://www.ncbi.nlm.nih.g
ov/pmc/articles/PMC2649121) .
PMID 19063604 (https://pubmed.ncbi.nlm.
nih.gov/19063604) .
7. Çelik S (January 2015). "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 (https://doi.o
rg/10.1016%2Fj.jim.2014.11.011) .
PMID 25435341 (https://pubmed.ncbi.nlm.
nih.gov/25435341) .
8. "Immunofluorescence Method" (http://ww
w.bio.davidson.edu/Courses/genomics/me
thod/IMF.html) . Davidson College.
 9. "Immunohistochemical Staining Methods"
(https://web.archive.org/web/2016080313
1150/http://www.dako.com/08002_03aug0
9_ihc_guidebook_5th_edition_chapter_10.p
df) (PDF). IHC Guidebook (Sixth ed.). Dako
Denmark A/S, An Agilent Technologies
Company. 2013. Archived from the original
(http://www.dako.com/08002_03aug09_ihc
_guidebook_5th_edition_chapter_10.pdf)
(PDF) on 2016-08-03. Retrieved 2014-05-14.

10. Fritschy JM, Härtig W (2001).

"Immunofluorescence" (http://www.els.net/
WileyCDA/ElsArticle/refId-a0001174.html) .
eLS. doi:10.1038/npg.els.0001174 (https://
doi.org/10.1038%2Fnpg.els.0001174) .
ISBN 047001590X.
11. Al-Mughales JA (2022). "Anti-Nuclear
Antibodies Patterns in Patients With
Systemic Lupus Erythematosus and Their
Correlation With Other Diagnostic
Immunological Parameters" (https://www.n
cbi.nlm.nih.gov/pmc/articles/PMC896409
0) . Front Immunol. 13: 850759.
doi:10.3389/fimmu.2022.850759 (https://d
oi.org/10.3389%2Ffimmu.2022.850759) .
PMC 8964090 (https://www.ncbi.nlm.nih.g
ov/pmc/articles/PMC8964090) .
PMID 35359932 (https://pubmed.ncbi.nlm.
nih.gov/35359932) .
Minor edits by Mikael Häggström, MD
- Attribution 4.0 International (CC BY 4.0)
license
12. Sonn GA, Behesnilian AS, Jiang ZK, Zettlitz
KA, Lepin EJ, Bentolila LA, et al. (March
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" (https://www.ncbi.
nlm.nih.gov/pmc/articles/PMC4794340) .
Clinical Cancer Research. 22 (6): 1403–
1412. doi:10.1158/1078-0432.CCR-15-0503
(https://doi.org/10.1158%2F1078-0432.CC
R-15-0503) . PMC 4794340 (https://www.n
cbi.nlm.nih.gov/pmc/articles/PMC479434
0) . PMID 26490315 (https://pubmed.ncbi.
nlm.nih.gov/26490315) .
13. Chalfie M (October 1995). "Green
fluorescent protein". Photochemistry and
Photobiology. 62 (4): 651–656.
doi:10.1111/j.1751-1097.1995.tb08712.x (h
ttps://doi.org/10.1111%2Fj.1751-1097.199
5.tb08712.x) . PMID 7480149 (https://pub
med.ncbi.nlm.nih.gov/7480149) .
S2CID 3944607 (https://api.semanticschola
r.org/CorpusID:3944607) .
14. Huang B, Bates M, Zhuang X (2009-06-02).
"Super-resolution fluorescence microscopy"
(https://www.ncbi.nlm.nih.gov/pmc/article
s/PMC2835776) . Annual Review of
Biochemistry. 78: 993–1016.
doi:10.1146/annurev.biochem.77.061906.0
92014 (https://doi.org/10.1146%2Fannurev.
biochem.77.061906.092014) .
PMC 2835776 (https://www.ncbi.nlm.nih.g
ov/pmc/articles/PMC2835776) .
PMID 19489737 (https://pubmed.ncbi.nlm.
nih.gov/19489737) .
15. Diaspro A, van Zandvoort MA (2016-11-03).
Super-resolution imaging in biomedicine.
CRC Press. ISBN 9781482244359.
OCLC 960719686 (https://www.worldcat.or
g/oclc/960719686) .
16. Leung BO, Chou KC (September 2011).
"Review of super-resolution fluorescence
microscopy for biology". Applied
Spectroscopy. 65 (9): 967–980.
Bibcode:2011ApSpe..65..967L (https://ui.ad
sabs.harvard.edu/abs/2011ApSpe..65..967
L) . doi:10.1366/11-06398 (https://doi.org/
10.1366%2F11-06398) . PMID 21929850 (h
ttps://pubmed.ncbi.nlm.nih.gov/2192985
0) .

External links
Images associated with autoimmune
diseases (http://www.ii.bham.ac.uk/clini
calimmunology/CISimagelibrary/) at
University of Birmingham
 Overview (http://www.bio.davidson.edu/
COURSES/genomics/method/IMF.html)
at Davidson College
Immunofluorescence (https://meshb.nl
m.nih.gov/record/ui?name=Immunofluo
rescence) at the U.S. National Library of
Medicine Medical Subject Headings
(MeSH)

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