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Antifade Reagents provide protection against fading or photobleaching for most common fluorophores used in live and fixed cell fluorescent imaging.
Photobleaching is a fundamental limitation of cell microscopy, reducing the number of photons emitted from a fluorophore molecule, and causing intermittent, fluctuating emission known as blinking. Blinking is caused when a fluorophore randomly enters transient, nonfluorescent states.
Effects of photobleaching can be moderated by reducing the intensity, frequency and duration of the excitation light. However, these steps limit the sensitivity of the experiment. Another approach is to increase the concentration of fluorophore being used, but this may result in cell toxicity.
Suppressing photobleaching using antifade reagents prolongs fluorescent signal intensity and longevity, allowing for high quality long exposure imaging experiments, which is especially important for time-lapse microscopy, super resolution microscopy (SRM) and signal molecule tracking microscopy, among others. Maximizing the amount of data achieved from an experiment, saves on time, lab resources and money.
The mechanism of action for antifade reagents is not fully understood, but they are proposed to work by scavenging the reactive oxygen species (ROS) produced during excitation of the fluorophores and so reducing oxidative stress damage in cells.
A popular cell-permeable antifade reagent, Trolox (Cat. No. 6002), a derivative of the natural antioxidant vitamin E, demonstrates an additional mechanism contributing to photobleaching protection. The Trolox anti-blinking and anti-bleaching effect is due to the generation of a Trolox quinoid derivatives, which forms in PBS buffer. The combination of Trolox and its oxidized form TX-quinone (TQ) produces a comprehensive antifade reagent, which works by triplet quenching through electron transfer, and the subsequent mopping-up of resulting radicals by the complementary redox reaction. This provides two-fold protection, reducing photobleaching at a quantum level, and reducing oxidative damage in cells. For more detailed information on the mechanism of Troxol and its protocol see: Cordes et al, 2009 On the mechanism of Trolox as antiblinking and antibleaching reagent. 131 5018. PMID 19301868.
Tocris offers the following scientific literature for Antifade Reagents to showcase our products. We invite you to request* your copy today!
*Please note that Tocris will only send literature to established scientific business / institute addresses.
This product guide provides a comprehensive list and background to the use of Fluorescent Probes and Dyes