Novel Multicolor Immunofluorescence Technique Using Primary Antibodies Raised in the Same Host Species

Simultaneous detection of multiple tissue antigens is one of the most frequently used immunohistochemical (IHC) techniques. In order to avoid cross-reactivity of each secondary antibody with multiple 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 that can be used with primary antibodies derived from a single host source. This technique includes modification of one primary antibody with biotin (ChromaLink™ Biotin) and a second primary antibody with DIG (ChromaLink™ Digoxigenin). For IHC staining, cells or tissue sections are incubated first with unconjugated primary antibody against the first target protein followed by detection with antiprimary secondary antibody conjugated to NorthernLights™ NL-637 tag (fluorescence in the far-red spectral region). Subsequently, the same tissue sections are incubated with a mixture of same species biotin-labeled primary antibody (against the second target protein) and DIG-labeled primary antibody (against the third target protein) followed by detection using a mixture of Streptavidin NorthernLights™ NL-493 tag (green fluorescence) and anti-DIG secondary antibody conjugated to a Rhodamine Red X™ tag (red fluorescence). This technique provides good 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 only primary antibodies raised in the same host species at their disposal.