Immunofluorescence (IF) utilizes antibodies labeled with a fluorescent molecule to localize and visualize proteins, glycans and small molecules in cells and tissues. IF is a great tool for scientists to study biological events. However, a key limitation of IF is the autofluorescence from intrinsic biomolecules.
Tissue autofluorescence often occurs from aldehyde fixation or from inherent native tissue components (such as collagen, elastin and red blood cells). The extent and intensity of autofluorescence background frequently make it difficult or impossible to distinguish specific signals from background especially in green and red channel fluorophores.
Quenching non-lipofuscin originated autofluorescence
Vector laboratories has developed a TrueVIEW Autofluorescence Quenching Kit which provides a novel way to diminish unwanted autofluorescence from non-lipofuscin sources and dramatically improve signal-to-noise ratio. The quenching action of the kit reagents provides a clear, unambiguous, “true view” localization of the target antigen.
Quenching Lipofuscin as a source of autofluorescence
Historically, Sudan Black or similar dyes has been used to tackle the autofluorescence challenges. Sudan Black is especially good at lowering autofluorescence due to lipofuscin (yellow-brown pigment granules composed of lipid-containing residues of lysosomal digestion). Lipofuscin is often a problem in human tissue samples and in aged animal tissue. The issue with Sudan Black is that it quenches a lot of the true positive fluorescent signal and is not very effective in red- and far-red channel.
Biotium has developed a lipofuscin quencher called TrueBlack, which tackles the issue in green and red channels and has a minimal reduction of the true fluorescent signal and it does not cause a high background.
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