Stepping Out of the Background
New innovations for reducing nonspecific fluorescence offer researchers clearer pictures and more multiplexing possibilities.
Many endogenously prevalent biomolecules, including collagens, hemoglobin, flavins, lipofuscin, and fatty acids, naturally autofluoresce. When performing microscopy, scientists must also contend with obfuscating signals introduced during the experimental workflow such as off-target antibody cross reactivity, antibody adsorption to the sample, and even the negative charges carried by fluorescent dyes that can promote nonspecific antibody binding. Researchers need to quench or filter out these nonspecific signals when performing immunofluorescence microscopy or staining so that they do not obscure the signal of interest.1
Scientists commonly combat nonspecific signals by using blocking and quenching agents. Blocking agents occupy potential binding sites for fluorescent dye-labeled primary and secondary antibodies, preventing them from attaching to unintended locations. However, while conventional blocking agents such as bovine serum albumin, gelatin, or casein can reduce nonspecific protein binding, they are not effective at blocking background from charged dyes. Quenching agents bind to autofluorescent biomolecules to reduce or absorb emission. However, they often emit fluorescence themselves in different portions of the spectrum.2
In light of these challenges, scientists are looking for new options to clear the way for more striking and effective staining, visualization, and imaging. One example of innovation in this area is the TrueBlack® IF Background Suppressor System, a set of buffers for immunofluorescence (IF) staining that is designed for blocking both nonspecific protein binding and background signal from charged dyes. These buffers can be used for both blocking and antibody dilution, and contain detergent for simultaneous blocking and permeabilizing in a single 10-minute step. Advances such as these offer flexible and effective solutions for countering nonspecific antibody binding and autofluorescence for scientists conducting a wide array of assays.