Biotium’s CF® Dye Tyramides enable flexible, low-cost 6-color multiplexing for immunofluorescence

Multiplex immunohistofluorescence (mIHF) has become an essential tool for studying complex tissue biology, enabling researchers to visualize multiple protein targets within a single sample while preserving spatial context. However, many existing multiplexing platforms remain costly, inflexible, or dependent on proprietary reagents, limiting accessibility for broader research applications. To address these challenges, open and scalable workflows are needed to make robust, reproducible, and cost-effective multiplex imaging more widely available to researchers.

In a 2026 protocol from the Journal of Microscopy, Riggi et al. created an open and flexible 6-color immunohistofluorescence (Flex-6 mIHF) workflow to investigate protein co-localization within the breast cancer tumor microenvironment. To overcome the background from autofluorescence in FFPE tissue sections that limits detection with IF, the protocol leveraged tyramide signal amplification (TSA) in combination with secondary antibodies conjugated to peroxidase-labeled polymers. This enabled robust signal enhancement and precise spatial resolution of biomarkers. The authors also outlined a stepwise validation strategy and essential controls to ensure reliable multiplex staining.

Within this workflow, CF® Dye Tyramides (Biotium) generated bright, covalently bound signals that can withstand repeated cycles of antibody stripping, facilitating sequential multiplexing without signal loss. This approach enabled simultaneous detection of up to six protein markers plus a nuclear stain in a single tissue section without requiring extensive image processing or spectral unmixing. By performing TSA with careful selection of antibodies, fluorophores, and order of target detection, the protocol produced high signal-to-noise images that could be directly analyzed, significantly reducing time and computational burden.

This protocol highlights how Biotium’s CF® Dye–based TSA reagents allow researchers to build flexible, high-performance multiplex immunofluorescence workflows without reliance on closed systems. By delivering exceptional brightness, photostability, and spectral diversity, Biotium’s fluorescent solutions help enable scalable, reproducible imaging protocols for cancer biology and beyond while making sophisticated multiplexing approaches more accessible to the broader scientific community.

Sequential multiplex tyramide labeling of human colon FFPE section with three CF® Dye Tyramides. Cytokeratin (pan) was labeled with CF®488A Tyramide (green); Histone H1 was labeled with Cyanine 555 Tyramide (red); ZO1 was labeled with CF®640R Tyramide (magenta). Credit: Biotium.

Learn more about Biotium’s products for TSA, including our TyraMax™ Amplification Dyes which offer improved brightness, photostability, and chemical stability over CF^® Dye Tyramides. Biotium also offers secondary antibodies conjugated to fluorophores or enzymes and a broad assortment of reagents for immunofluorescence microscopy.

Full Citation:

Riggi, J. A. M., Daumerie, A., Benhaddi, N., Berlière, M., Galant, C., González-Antelo, A., Nana, F. A., Van Bockstal, M. R., & Bouzin, C. (2026). A detailed protocol for open and low-cost six-plex immunofluorescence (Flex-6 mIHF) with a proof-of-concept study on breast cancer tissue. Journal of Microscopy, 1–19. https://doi.org/10.1111/jmi.70068