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Immunofluorescence - Types, Techniques, and Limitations

Posted by Fred Koenig on

Dec 15th 2020

Immunofluorescence - Types, Techniques, and

Limitations

What is immunofluorescence?
Immunofluorescence is one of the techniques used within light microscopy, especially on
microbiological samples. It
uses
a fluorescence microscope
to observe antibodies, bonded to their
antigens, with fluorescent dyes attached to
specific biomolecule targets. Observers can therefore
visualize the distribution of target molecules throughout a
sample. The specific antigen region that
an antibody recognizes is called an epitope. Since many antibodies can
attach to the same epitope,
and there is variance between the levels of binding between antibodies and the epitopes
they can
recognize, efforts are being made to further develop and improve epitope mapping. The binding of a
fluorophore to an antibody also cannot interfere with the immunological specificity of the antibody
or with the
binding capacity of its antigen.

Using antibodies to stain proteins is called “immunostaining,” and immunofluorescence is a

commonly cited example of
such a technique. Taking advantage of the antibody-antigen
relationship in tissues has led to a field called
immunohistochemistry. In this field, practitioners use
fluorophores to visualize the location of the antibodies.

Immunofluorescence can be used to analyze the distribution of proteins, glycans and small
molecules, both biological
and non-biological. It can be used to visualize a wide range of samples,
from intermediate-sized filaments to tissue
sections, individual cells, or cultured cell lines.

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Epitope insertion can be used with immunofluorescence to determine structures, in cases where the
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topology of a cell
membrane is as yet undetermined.
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Another use of immunofluorescence is as a semi-quantitative method of gaining insight into levels
and localization
patterns of DNA methylation. There is some subjectivity in the analysis of the
methylation levels, and the method is
more time consuming than true quantitative methods.

Immunofluorescence can also be used in fluorescence staining in combination with some non-
antibody methods, such as
DAPI in the labelling of DNA.

The simplest microscope design used for examples of immunofluorescence samples is the
epifluorescence microscope.
Another common design is the confocal microscope.

Also commonly used are various designs of super-resolution

microscopes which are capable of
much higher resolution levels.

Tagging
Fluorochrome-labelled antibodies are made by conjugating a fluorochrome to the antibody, a
process also known as
tagging. A process called fluorescent antigen technique can also be used. In
this process, an antigen is conjugated
to the antibody with a fluorescent probe. Fixed antigen in the
cytoplasm and cell surface antigens on living cells
can both be stained via such procedures, which
is known as “membrane immunofluorescence.” The complement of the
antibody-antigen complex
can also be labelled, or tagged, with a fluorescent probe.

Types of Immunofluorescence
Immunofluorescence techniques fall into two general classes: Primary, also known as direct; and
secondary, also
called indirect.

1. Primary (direct)

Primary immunofluorescence is a process which includes chemically linking a single, primary

antibody to a
fluorophore.

The primary antibody binds to a specific region of the target molecule. The region is called the
epitope. The immune
response system of an organism with adaptive immunity is thereby
manipulated. Fluorescent microscopy can detect the
attached fluorophore. The fluorophore,
depending on the messenger used, is excited and emits a specific wavelength
of light.

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This technique is a little less common than the secondary,
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or indirect technique, but nonetheless has
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some
advantages. Because the messenger attaches directly to the antibody (hence the alternate
name “direct”), there is
less possibility
What of antibody
can we helpcross-reactivity,
you find? there are fewer steps in the
procedure, which makes it faster and
less likely to contain errors, and there is less non-specific
background signal.

There are downsides too though. The number of fluorescent molecules that are able to bind to a
primary antibody is
limited. This makes the direct method significantly less sensitive than its indirect
counterpart, and false
negatives may occur. There is also the cost factor. Primary antibody is
expensive, sometimes up to $400.00/mL, and
the direct method uses a lot more of it than the
indirect method does.

2. Secondary (indirect)

Secondary immunofluorescence, also called indirect immunofluorescence, uses two antibodies.

The first one (primary) is not labelled, and specifically binds the target molecule. The secondary
antibody is the
one that carries the fluorophore.

It recognizes the primary antibody and binds to it. A single primary antibody can be bound by
multiple secondary
antibodies, thereby providing an amplified signal because there are more
fluorophore molecules per antigen. This
process entails more steps than primary/direct methods
and therefore takes more time. It is more flexible, however,
as different secondary antibodies can be
used, and more detection techniques are applicable, for a single type of
primary antibody.

This process is based on the idea that we can divide each antibody into two theoretical parts, a
variable region and
a constant region. The first recognizes the antigen, and the second makes up the
structure of the antibody molecule.
The molecule is, in reality, composed of four polypeptide chains.
Two of these are heavy chains, and two are light.
Several primary antibodies can have different
variable regions – meaning that they can recognize various antigens –
while all sharing the same
constant region. A single secondary antibody can therefore recognize all of them, so
there is no
need for the trouble or cost of modifying the primary antibodies to directly carry a fluorophore.

Raising the antibody in different species produces different primary antibodies (thought this is not
the only method,
it is the usual one). Primary antibodies from a goat might be combined with dye-
coupled rabbit secondary antibodies
that recognize the constant region of the goat antibody,
creating “rabbit -anti-goat” antibodies. A second set might
then be made from primary antibodies in
a mouse that could be recognized by a separate “donkey anti-mouse” secondary
antibody. Since the
dye-coupled antibodies are difficult to make, and this process allows reuse of them in multiple
experiments, the cost is reduced over several experiments.
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Limitations
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Photobleaching can be a significant

What problem
can weinhelp
immunofluorescence
you find? microscopy. Photobleaching
can reduce the activity
in a sample, simultaneously reducing observable data. It can be mitigated by
limiting the overall light exposure,
either by reducing intensity, or time span; by increasing the ratio of
fluorophores employed; or by using
fluorophores that are known to be less prone to bleaching.
Examples of more robust fluorophores include Alexa
Flours, Seta Fluors, and DyLight Fluors.

Autofluorescence can also be a problem. Undesired extraneous fluorescence (both specific and
nonspecific) can be
emitted from the sample tissue or cell, when a targeted antigen contains
antigenic contaminants (specific
fluorescence), or when a fluorophore improperly fixates, or a
specimen is dried out, causing a loss in the probe’s
specificity (nonspecific fluorescence).

Immunofluorescence is limited to fixed, or “dead” cells when the goal is to observe structures within
the cell. This
is because the antibodies do not penetrate the cell membrane when reacting with
fluorescent labels. The site of
natural localization inside the cell must be accessible for the
antigenic material to properly affix to it. Cancer
cells are often too small for some intact antibodies
to dye them in vivo. This results in slow tumor penetration and
a longer circulating half-life, though
some research in the area of diabody use may help us to get around these
problems. Binding
proteins in the supernatant, or on the exterior of a cell membrane, can allow even living cells to
be
stained. Some proteins of interest might become cross-linked, though, depending on the fixative
employed, and
non-specific binding could result, giving false negative or false positive signals.

Another way to go about this is to use recombinant proteins that contain fluorescent protein
domains such as green
fluorescent protein, or GFP. These tagged proteins show their localization
within live cells. Though this technique
solves some scientific issues, it triggers others. Once the
cells are transfected or transduced with the GFP tag,
they become at least S1 or above organisms,
and therefore require stricter security standards in the lab. The
technique alters the genetic
information of the cells.

New York Microscope Company has a wide range of microscopes

for sale online, with international
shipping from our location in the USA. Contact us today for all things
fluorescence microscopes, or
browse more informative articles below:

Fluorescence microscopy
What is a compound
microscope?
What is a stereo microscope?
Stereo vs compound
microscopes

Sources
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https://www.bio-rad-antibodies.com/flow-cytometry-which-fluorochromes-are-useful.html

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https://rupress.org/jem/article/168/1/107/49566/Lipooligosaccharides-LOS-of-Neisseria-
gonorrhoeae
What can we help you find?
https://www.bio.davidson.edu/Courses/genomics/method/IMF.html

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