Technical Notes

How to reduce and remove autofluorescence

Multiplex immunofluorescence (mIF) is a powerful technique for visualizing multiple biomarkers in the tumor microenvironment. Antibodies, labeled with fluorescent molecules, are used to detect the antigen-antibody complexes. However, tissues have intrinsic autofluorescence from biological components that can interfere with the measurement of markers of interests in mIF assays. Autofluorescence is a general term describing the background fluorescence in tissue sections unrelated to the specific signal generated during an immunofluorescent assay. Tissue components, such as proteins, lipids, and red blood cells, absorb and then emit light upon exposure with the microscope’s light source. In addition, extrinsic tissue components, such as formalin used during tissue fixation method, can increase tissue autofluorescence.

Autofluorescence affects the quality of the staining and reduces the accuracy of signal detection and quantification. Thus, when performing mIF assays, it is important to reduce autofluorescence to achieve high-quality stainings that allow clear and unambiguous analysis of the biomarkers.

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