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 visualisation of the
distribution of the target molecule through the sample.
Immunofluorescence is a widely used example of immunostaining and is a specific example
of immunohistochemistry that makes use of fluorophores to visualise the location of
the antibodies.
Immunofluorescence can be used on tissue sections, cultured cell lines, or individual cells,
and may be used to analyse the distribution of proteins, glycans, and small biological
and non-biological molecules.
Immunofluoresence 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.
Types of immunofluorescence
There are two classes of immunofluorescence techniques, primary and secondary.
Primary Primary, or direct, immunofluorescence uses
a single, primary antibody, chemically linked to a fluorophore.
The primary antibody recognizes the target molecule 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.
Direct Immunofluorescence, although somewhat less common, has notable advantages over the
secondary 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.
This also limits the possibility of antibody cross-reactivty and possible mistakes throughout
the process.
However, 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
Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first antibody
specifically binds the target molecule, and the secondary antibody, which carries the
fluorophore, recognises 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.
This protocol is more complex and time consuming than the primary protocol above, but it allows
more flexibility because a variety of different secondary antibodies and detection techniques
can be used for a given primary antibody.
This protocol is possible because an antibody consists of two parts, a variable region and
constant region.
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,
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.
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 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.
Immunofluorescence is only limited to fixed cells when structures within the cell are
to be visualized because antibodies cannot cross the cell membrane.
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.
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.
See also Immunochemistry
Cutaneous conditions with immunofluorescence findings
References
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 SynD - Automatic synapse and neurite detection
in immuno-fluorescence images