It is easy to see why researchers have employed immunofluorescent readouts for decades. The method offers the advantage of superior multiplexing and easier identification of co-localised molecules. Furthermore, it also provides a dynamic range that affords visualisation of both high- and low-abundance targets within the same sample. The need to detect an ever-growing number of parameters from increasingly complex sample material is driving the evolution of highly sophisticated immunofluorescent reagents for research. To keep up with customer demand, manufacturers are constantly developing innovative products. Whether it is to streamline workflows, enhance data quality or extend the capabilities of this highly popular detection method.
Irrespective of the chosen application, an immunofluorescent workflow typically includes sample incubation with a target-specific primary antibody. This is then followed by the addition of a fluorescently labelled secondary antibody for visualisation. However, while this tried and tested method is used routinely in laboratories worldwide, many researchers are realising the benefits of incorporating signal amplification techniques. This is because, this technique could have previously unseen benefits such as better at detecting low-abundance targets.
One method of amplifying fluorescent signals is to exploit the binding affinity of biotin for streptavidin in a multi-layered approach. For example, using a biotinylated secondary antibody for detection. It is possible to accumulate a greater number of fluorophores at the target site through the addition of streptavidin followed by a biotinylated anti-streptavidin antibody and a fluorescent streptavidin conjugate.
An alternative, biotin-free, method is to add a bridging antibody between the target-specific primary antibody and the fluorescently-labelled detection antibody. This is a technique exemplified by Vector Laboratories’ VectaFluor™ Excel Amplified Staining System. Using a high-affinity, unconjugated anti-species antibody produced in goat followed by addition of a VectaFluor™ DyLight® dye-conjugated anti-goat IgG antibody, substantial increases in sensitivity have been achieved.
Better assay windows
Although a strong fluorescent signal is usually regarded as being indicative of successful staining, bright fluorescence is considerably more meaningful when combined with minimal background signal. Background signal most commonly arise from autofluorescence of the sample material, media or culture vessels, or occurring due to non-specific antibody binding. Moreover, researchers are acutely aware of the importance of reducing background signal by thoroughly optimising staining protocols. This includes evaluation of blocking and washing steps, titration of antibody reagents, and careful selection of fluorescent dyes.
Vector Laboratories have developed their novel Vector® TrueVIEW™ Autofluorescence Quenching Kit to address the problem of unwanted fluorescence occurring in tissue sections due to aldehyde fixation, the presence of red blood cells, or from structural elements such as collagen and elastin. The kit employs a simple one-step method for easy incorporation into existing staining protocols. Furthermore, they are designed to retain specific fluorescent antigen staining while reducing autofluorescence.
One of the many strengths of immunofluorescent staining is that it facilitates the long-term storage of processed slides. However fluorescent signals can fade significantly over time. This occurs due to photobleaching, the process by which fluorophores undergo photochemical alteration following exposure to light. Although researchers are aware that the easiest way to minimise photobleaching is to keep exposure times as short as possible, this can be impractical when dealing with low abundance targets.
By developing dyes that are more resistant to photobleaching, manufacturers have gone some way toward addressing the issue of fading. Another approach has been to produce fade-resistant mounting medias. One of the latest antifade products to reach the market, Vector Laboratories’ VECTASHIELD® Vibrance™ Antifade Mounting Media offers excellent protection against fading across the visible spectrum, even under far-red wavelengths. In addition, it also allow same-day viewing without compromising on signal intensity or retention.
The approaches described here represent just some of the many inventive technologies employed to take immunofluorescent detection to new levels. Advances in instrumentation and data handling will also be essential as researchers will demand more information from precious sample material. With its scope continuing to grow, immunofluorescent detection has an extremely bright future.
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