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Multi-color Fluorescence Immunohistochemistry Using Primary Antibodies Raised in the Same Host Species

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Tutorial Video Abstract


Simultaneous detection of multiple tissue antigens is one of the most frequently used immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of secondary antibodies with unwanted primary antibodies when doing either dual- or triple-labeling immunofluorescence it is necessary to use primary antibodies raised in different host species such as mouse, rabbit and goat. However, in many cases suitable primary antibodies raised in different species are unavailable. We have developed a novel technique for triple-labeling immunofluorescence IHC that can be used with primary antibodies derived from a single host source. This technique includes modification of primary antibodies by conjugating them to Biotin and Digoxigenin followed by incubation with NorthernLightsTM fluorescent secondary antibodies (R&D Systems, Inc.).

This novel technique provides an excellent spectral separation of colors depicting different antigens of interest while avoiding cross-reactivity between irrelevant primary and secondary antibodies. In addition, this multiplexed IHC technique provides significant convenience to researchers who have at their disposal only primary antibodies raised in the same host species.

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