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Multicolor immunofluorescence detection of immune markers in FFPE tumor samples

Immunotherapy is emerging as one of the most promising treatment options for cancer as demonstrated by the awarding of this year’s Nobel Prize in Medicine to two immune oncology pioneers. However, as with other cancer therapies, it appears to benefit only a small proportion of individuals. High-throughput multiplex immunophenotyping of tumor samples for clinically relevant prognostic biomarkers can greatly improve treatment outcomes.

In a recent publication in the Journal of Pathology, the authors Ijsselsteijn et al. describe a rapid and efficient 7-colour immunofluorescence (IF) method for extensive phenotyping of FFPE tumor samples by multispectral fluorescence microscopy. Using a combination of indirect and direct IF, the protocol improves upon other widely-used techniques such as multiplex immunodetection by tyramide signal amplification (TSA). It overcomes limits such as the number of targets that can be detected simultaneously, lack of spatial context, cumbersome workflows, and/ or compromise to tissue integrity and morphology. The system is also flexible and can easily be combined with TSA-based detection of low-abundance antigens. This technique can serve as a high-throughput, diagnostic tool in clinical analysis, or be used a complementary method for the validation of discovery sets generated by RNA sequencing or mass cytometry.

Learn more about Biotium’s bright and photostable CF® dyes, CF® dye-labeled primary and secondary antibodies, and Mix-n-stain™ CF® dye antibody labeling kits for indirect and direct immunofluorescence applications.


MCF-7 cells stained with rabbit anti-COXIV and CF®555 goat anti-rabbit (mitochondria, red) and mouse anti-ZO1 and CF®647 goat anti-mouse (tight junctions, green). Mounted in EverBrite™ Mounting Medium with DAPI (nuclei, blue).