What is immunofluorescence?

Immunofluorescence is the process by which an antigen is detected with an antibody and is then visualized using a fluorophore. The fluorescent readout is generated by using a light source to excite the fluorophore, which then produces a transient light emission as it returns to its ground state. The emitted light has a higher wavelength than that which was used for excitation, and is detected with a specialized reader. Immunofluorescence can be used as the detection method in a wide variety of laboratory applications, including Western blotting, immunocytochemistry, immunohistochemistry, flow cytometry, ELISA and lateral flow.

A huge range of fluorophores is commercially available, and includes fluorescent dyes, fluorescent proteins and lanthanides. Each fluorophore will have a unique set of properties, all of which should be taken in to account when selecting a suitable label for the downstream application. The maximal absorption and maximal emission wavelengths should be considered, in addition to the extinction co-efficient and the Stokes shift. The extinction coefficient defines how well a fluorophore absorbs light at a particular wavelength; fluorophores with high extinction coefficients are generally brighter. The Stokes shift is the difference between the maximum absorbance and emission wavelengths of a fluorophore; a greater Stokes shift is often desirable since it is indicative of less overlap between the two wavelengths.

Immunofluorescence offers several advantages over other methods of detection:

✓ Multiplexing is possible – saves time, money and precious sample
✓ High sensitivity
✓ Quantitative
✓ Signal stability

Direct versus indirect detection

Like any method of immunodetection, immunofluorescence can be either direct or indirect. Direct detection relies on the use of labeled primary antibodies, while indirect detection utilizes labeled secondary antibodies.

Direct vs Indirect detectionDirect versus indirect detection. Direct detection (left) relies on the use of labeled primary antibodies. Indirect detection (right) requires labeled secondary antibodies.

direct vs indirect detection - immunofluorescence

Non-specific binding of labeled secondary antibodies is a common problem during immunodetection. It can occur if the labeled secondary antibodies cross-react with endogenous immunoglobulins on the tissues or cells, or with immunoglobulins in the antibody diluent, and can also result from cross-species reactivity with other labeled secondary antibodies. Direct detection therefore offers several clear advantages over indirect methods:

✓ Non-specific binding is avoided since secondary antibodies are not used
✓ Multiplexing is possible with antibodies from the same species
✓ Faster since there is no secondary antibody incubation step and therefore fewer wash steps
✓ Data quality is improved through assay simplification

Despite the benefits of direct detection, many immunoassays still rely on the use of labeled secondary antibodies. The main reason for this is that direct labeling of primary antibodies with detection moieties such as fluorescent dyes can be a complicated and time-consuming process, which requires specialist knowledge.

Lightning-Link® for direct antibody labeling

Lightning-Link® from Expedeon is an innovative technology that enables direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups. The product range includes kits for labeling antibodies with a wide range of fluorophores, covering the spectrum from UV to far infra-red, and is therefore ideally suited for applications which rely on an immunofluorescent readout. Many commercial antibody sources often do not offer the required antibody conjugated to the desired label, however Lightning-Link® enables the end user to quickly and easily generate a panel of labeled antibodies for immunofluorescence using commercially-sourced unlabeled antibodies.

Lightning-Link process diagram

The Lightning-Link® conjugation process.

The benefits of Lightning-Link® include:

✓ Quick and easy to use
✓ Requires only 30 seconds hands-on time
✓ No separation steps involved so 100% of the antibody or protein is recovered
✓ Possibility to label from as little as 10ug to a gram or more of antibody

To find out more, why not watch our video?

Immunofluorescence data generated using Lightning-Link®.  RAW264 macrophage cell line stained with chicken anti-F4/80 antibody conjugated to fluorescein using Lightning-Link®, counter-stained with DAPI.


Tandem dyes to increase the number of assay readouts

Our Lightning-Link® product range includes several tandem dye labels, consisting of a donor fluorophore and an acceptor fluorophore which have been covalently attached to one another. The use of tandem dyes is a very easy way to increase the quantity of assay readouts. If, for example, the number of excitation lasers is a limiting factor in a flow cytometry experiment, the inclusion of antibodies which have been directly labeled with tandem dyes can be used to generate multiple outputs. For instance, DyLight® 488, R-phycoerythrin (PE), PerCP/Cy5.5 and PE/Atto 594 can all be excited using a 488nm laser, yet generate green, yellow, red and infra-red emissions respectively, each of which can be measured using a different detector.

Lightning-Link® tandem dye labeling kits

PE/Texas Red®

To find out more about the advantages of working with tandem dyes, why not read our blog?


Europium conjugation kits

Our europium conjugation kits contain specially-treated 200nm europium beads, which become covalently bound to the antibody or protein via free amine groups to form highly stable conjugates. This unique product to Expedeon can be employed in either an immunochromatographic lateral flow assay or a microwell-based assay by using an appropriate reader (detection of europium conjugates in a lateral flow assay requires either a fluorescence strip reader or a UV transilluminator, while a microplate assay will require a plate reader which is fitted with the appropriate filters).

Our europium conjugation kits provide several advantages:

✓ Quick and easy to use – the conjugate is ready to use in just 35 minutes
✓ 15-fold higher sensitivity compared with other common particles
✓ Specially treated particles are resistant to aggregation and require no extensive pH optimization

Lateral flow assay using our specially-treated europium beads. The europium beads were conjugated to a monoclonal anti-CRP antibody, and used to quantify a titration of CRP. The conjugate is easily able to detect as little as 63pg/ml CRP, in part due to the low background and lack of aggregation.

Click to read more

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**This webinar was produced by Innova Biosciences which is now fully integrated with their company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   Dr Nick Gee, the CEO and CSO of Expedeon, explains the benefits of using directly conjugated antibodies and the various approaches and techniques…

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