Immunofluorescence (If)

Immunofluorescence (IF) is a technique that permits visualization of virtually many components in any
given tissue or cell type. This broad capability is achieved through combinations of specific antibodies
tagged with fluorophores.
➢ It can be performed on cultured cells or cell suspensions, as well as on specific targets in tissues
samples or entire organisms.
➢ Fresh samples can also be used in IF studies if they are snap frozen or placed in Michel’s
transport medium, which allows transportation and storage at room temperature for up to 72
hours.
Steps:
1. Fixation: is an essential preliminary step in IF staining in order to prevent autolysis,
mitigate putrefaction, and preserve morphology while maintaining antigenicity.

❖ The ideal fixation method serves to immobilize target antigens without disturbing cellular
architecture to allow antibodies maximum access to any targeted cellular components. It is
possible that a given fixative may adequately preserve the immunoreactivity of a particular
epitope, while degrading or masking other epitopes within the same protein.
❖ Consequently, as there is no universal fixative for every antigen, optimal fixatives and fixation
methods may need to be determined empirically based on the given antigen and sample type.
❖ Chemical fixatives include cross-linking reagents and organic solvents, used separately or in
combination:
a) Cross-linking (additive) reagents bind to cellular and tissue components by addition,
forming intra and intermolecular methylene cross-links. Common examples include
formaldehyde and glutaraldehyde.
b) Organic solvents work to remove lipids and dehydrate cells, denaturing and precipitating
cellular components. In addition, by permeabilizing cell membranes, organic solvents
eliminate the need for detergent treatments. Common examples include methanol and
acetone.
Following fixation, tissues are often embedded into paraffin in order to solidify the sample
for sectioning.
• Thin slices embedded in a hard matrix allow for dyes, probes, and antibodies to
effective reach target sites without the obstruction of multiple cell layers.

• Paraffin blocks can be stored at room temperature, but they must be kept in the dark
under moisture controlled conditions.

• Embedded tissue blocks should be considered infectious material, ensuring all proper
personal protective equipment is worn when handling or working with blocks.

• Once paraffin sections have been cut and mounted onto glass slides, however, they
must be deparaffinated and rehydrated so that only the tissue section remains on the
slide for subsequent antigen retrieval and IF procedures.
• Xylene is the most common medium for deparaffinization, followed with washes of
ethanol and distilled water for rehydration.

1|Page
 Immunofluorescence (If)
2. Antigen retrieval: is necessary to restore epitope-antibody reactivity. Reactivity is
altered during fixation, in which proteins undergo structural modification, forming cross-links
that can mask the target epitope. This is especially prevalent with the use of additive reagents.

The need for antigen retrieval is dictated by various factors: target antigen identity, antibody
character, tissue type, the method and duration of fixation.

Antigen demonstration levels can be recovered using retrieval agents to cleave the cross-links
formed during fixation.

There are two main methods of antigen retrieval: Protease-Induced Epitope Retrieval (PIER)
and Heat-Induced Epitope Retrieval (HIER). As with any technique, both methods must be
optimized prior to any application.

In PIER, enzymes serve as the primary method in restoring antigenicity. Though the exact
mechanism is not yet fully understood, it is generally thought that the enzymes cleave the
protein cross-links to unmask the target epitopes. Common examples of proteases include
Proteinase K, Trypsin, Pepsin, and Pronase.

The specific enzyme to be used should be detailed in any antibody data sheet provided by the
manufacturer. However, there are several disadvantages to the PIER method.

Because the enzymes used for PIER cannot target only the exact protein cross-
links present in the fixed tissue sample, non-specific enzyme digestion can
potentially destroy tissue morphology and any antigens of interest.

Consequently, strict incubation times and optimal enzyme concentrations must be

determined beforehand. Much higher rates of restoring immunoreactivity can be achieved
with the HIER method.

With HIER, heat and pressure are used to restore antigenicity. Just as in the case of PIER,
the exact mechanism of reversing protein cross-links is unknown, though it is assumed
secondary and tertiary protein structure must be restored for the epitope to be unmasked.

In general, the HIER method involves heating the mounted tissue sample in a buffer solution,
with heat to cleave cross-links and buffer to maintain protein conformation. Buffer solutions
can be categorized into low, neutral, and high pH solution compositions.

Low pH solutions are usually buffered with glycine-HCl, neutral pH solutions with citric acid,
and high pH solutions with Tris or EDTA. Though high pH solutions usually serve as the most
effective buffers, high pH and EDTA have higher chances of convoluting tissue and cellular
Morphology.

2|Page
 Immunofluorescence (If)
As different HIER methods vary upon combinations of appliance type, temperature, duration,
and pH, the optimal method and specifications must be determined by conducting preliminary
control studies.

In addition, an HIER device should be chosen based on the following factors: temperature
range, buffer volume, minimal evaporation, and minimal boil-over.

Following fixation and antigen retrieval, either of two IF methods can

be employed: Direct(Primary) IF or Indirect (Secondary) IF.
The direct method The indirect method
fluorophore label is conjugated directly to the involves a two-step incubation process: 1) a
primary antibody that will be reacting with primary antibody binds to the target epitope,
the target epitope. 2) a fluorophore-tagged secondary antibody
recognizes and binds to the primary antibody
quicker
more widely employed for its high sensitivity,
signal amplification, and its ability to detect
several targets in the same sample.

To prevent the secondary antibody from cross-reacting with endogenous immunoglobulins in

the tissue sample, the primary antibody should be derived from a different species than that
of the sample. Consequently, the secondary antibody must be against the host species of the
primary antibody.

Commonly, secondary antibodies are conjugated with fluorescent labels which emit upon
photoexcitation. Enzymatic labels can also be conjugated to react with chromogen substrates for
colorimetric analyses. For greater signal amplification, polyclonal or biotinylated secondary
antibodies can be employed. For each primary antibody, polyclonal secondary
antibodies are able to recognize multiple epitopes to increase binding and signal levels.

Similarly, multiple fluorochrome-protein (avidin or streptavidin) complexes can bind to a

single biotinylated secondary antibody to increase signal levels. A combination of these
methods can be used as well for greater signal amplification, as detailed later on.
3. Blocking: Before any antibody application, however, blocking must be performed on
tissue samples to prevent antibodies from binding to non-target epitopes
present. Blocking reagents should be chosen to ideally have no affinity for the target
epitopes, high binding rates to non-target reactive sites, and stabilization of cellular
morphology. As no universal methodology exists, the best combination of blocking reagents,
blocking duration, and antibody types must be determined empirically.
In general, most blocking buffers fall into the following categories: protein solutions, normal
serums, protein-free commercial buffers. Protein blocking solutions are concentrated protein
buffers that essentially bind all proteins present in the sample. As antibodies are forced to
compete with the blocking protein for target epitopes, non-specific binding can be reduced.

3|Page
 Immunofluorescence (If)
Common examples include bovine serum albumin (BSA), non-fat dry
milk, and gelatin.

Normal serum is a blocking reagent containing antibodies from the same species as that of
the secondary antibody. This property serves to block non-target reactive
sites to which secondary antibodies would otherwise bind.
Finally, protein-free commercial blocking buffers are also widely available, with multiple
options specific for various antibodies as well as increased shelf life.

4. Fluorescence detection: is possible with use of fluorophore-conjugated antibodies, which

emit light upon excitation via light of a shorter wavelength.

• The most commonly used fluorophores are fluorescein isothiocyanate (FITC) and
tetramethyl rhodamine isothiocyanate (TRITC).

• The ideal fluorophore for a particular experiment can be determined by various factors.
Depending on the use of confocal or epifluorescence microscopes, fluorophores that can
actually be excited and detected by the equipment available must be selected.

• In addition, most manufacturers include fluorophore reference charts indicating

maximum excitation and emission wavelengths of the given fluorophore.

• High extinction coefficients and high quantum yields are also important factors in
choosing the ideal fluorophore.
• With poor extinction coefficients and quantum yields, the fluorophore will appear very
dim.Fluorophores are also susceptible to photobleaching.

• To minimize photobleaching, photostable fluorophores can be selected, excitation

duration and intensity can be reduced, and antifade mounting reagents can be applied.
When multiple fluorophores are in use, additional issues arise.

• Spectral overlap occurs when the excitation and emission wavelength spectrum of one
fluorophore includes the spectrum of the other fluorophore in use. Selected
fluorophores should be checked for spectral overlap in reference to the manufacturer’s’
spectrum viewer.

• When there exists contrasting expression levels of the different antigens of interest,
dimmer fluorophores should be used to detect abundant antigens, with sparse
antigens detected by brighter fluorophores.

4|Page
 Immunofluorescence (If)
The immunofluorescence method has inherent advantages with regards to signal
amplification, targeting specificity, resolution, and analytical capabilities. The IF method can
be further improved for greater amplification of signal with the use of polyclonal antibodies in
conjugation with enzyme complexes.

One such example is the fluorophore-

labeled Streptavidin-Biotin complex. Being polyclonal, multiple biotinylated secondary

antibodies are able to recognize and bind to a single primary antibody. In turn, binding of
greater numbers of fluorophore-labeled Streptavidin per primary antibody greatly amplifies
the fluorescence signal relative to a simple direct IF method. In addition, IF methods allow
the possibility of multiplexing.

For chromogen labeling, target antigens in close proximity will be masked due to
counterstaining. However, multiple antigens regardless of spatial orientation can be stained
simultaneously since different fluorophores are onlysensitive to their corresponding excitation
wavelengths.
This multiplexity is ideal for co-localization studies requiring high-resolution multi-antigen
imaging. Furthermore, for fluorescence detection, better image qualities and quantitative
microscopy are available.

Confocal fluorescent microscopes can obtain higher resolutions and multi-planar images,
and fluorescent detection methods avoid “fuzzy” images resulting from chromogenic
enzyme precipitates in imaging protein localization.

While the enzymatic approach of chromogenic methods limits quantitative capabilities of

immunochemical analyses, fluorescent probes permit high-throughput and quantitative
automated approaches.

5|Page