Autofluorescence has been a recurrent problem in immunofluorescent staining. It impedes on distinguishing specific signaling vs. non-specific (background) in tissues that contain natural fluorescence from endogenous fluorophores. Collagen, elastin, flavins, red blood cells and lipofuscins are known to have natural endogenous fluorescence. A tissue that contains any of the above components in images are prone to have miniature speckles that have a strong fluorescent excitation, anywhere between 360nm to 647nm. Autofluorescence can also hinder data analysis by making it difficult to co-localize proteins due to specific signaling being masked. Fortunately, several autofluorescence quenchers have been developed to bypass unwanted non-specific signaling from endogenous fluorescence.
What Is Autofluorescence Quenching?
Autofluorescence quenching is the process in which the intensity of emitted light from fluorescent molecules is decreased. Three common quencher kits discussed today will be TrueVIEW, TrueBlack and Sudan Black b.
- TrueVIEW diminishes unwanted autofluorescence from non-lipofuscin sources (collagen, elastin and red blood cells) in tissue sections that have an aldehyde-based fixative. It can be stored at room temperature and/or in the fridge for 48 hours and should be discarded after. TrueVIEW will stain tissue sections blue, but it should not interfere with immunofluorescence labeling. It is most effective in kidney and spleen tissue.
- TrueBlack is primarily used to quench lipofuscin autofluorescence. It can improve background from other sources, such as collagen, elastin, and red blood cells. It can be stored at room temperature in a dark place for 12 months. It is most effective in human brain and retina tissue.
- Sudan Black b, also known as Solvent Black 3, acts as a dark mask to diminish lipofuscin autofluorescence and other sources without covering immunofluorescence labeling. It comes as a powder so it can be stored at room temperature long-term. It is known to be effective in multiple tissue types, such as pancreas, kidney, brain, mandibles, etc.
All three common quenchers must avoid any washes that contain detergent, as it will wash the dye away. Autofluorescent quenchers have enhanced immunofluorescent staining, allowing Visikol to visualize specific proteins and structure.
What Does Visikol Use for Autofluorescent Quenching?
At Visikol, we use Sudan Black b as our autofluorescent quencher. We prepare it in 0.3% of the Sudan Black b powder in 70% ethanol overnight on a shaker in the dark. The next morning, it is filtered and ready for use. Incubation is done after immunolabeling for about 10-15 minutes depending on the tissue type. Visikol has chosen to use Sudan Black b due to documented validation of multiple tissue types and it’s compatibility with our multiplexing and striping protocols. Visikol works closely with clients to help them meet their drug discovery goals. Reach out to us today to chat with our team if you have any questions or want to know more about our services.
Without Sudan Black b
With Sudan Black b