The high throughput immunofluorescence technique known as multiplex immunofluorescence (mIF), which is based on multispectral imaging and tyramide signal amplification, enables the simultaneous detection of many markers on a single tissue segment without damaging the tissue architecture.

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Crucial elements

Using multispectral photography to produce pictures for analysis, multiplex immunofluorescence is a method based on enzyme-based tyramide signal amplification.

After staining a slide, multiplex immunofluorescence requires that all of the fluorophores be excited; however, the emission spectral range and the excitation range cannot overlap.

Linear unmixing of pictures, tissue segmentation into tumor and stroma, individual cell segmentation, and marker phenotyping based on marker of interest are the fundamental principles of data processing.

The location of individual cells within a tissue may be determined using subsequent data, which can be used to create various datasets detailing cell characteristics and nearest neighbor distances.

What is immunofluorescence microscopy using multiplexing?

With the use of fluorescent markers and various light wavelengths, multiplex immunofluorescence microscopy is a state-of-the-art technology that may be used to answer a wide range of research questions by producing complex multi-spectral pictures. Inventing easily available tissue clearing reagents for multiplex immunofluorescent 3D microscopy, Icuradx has led the way in cell viewing since its founding.

Imperative Microscopy

In fluorescence microscopy, certain sites of interest may be seen and quantitatively evaluated by the binding of a dye-tagged antibody to the target antigen. This target appears as a fluorescent pattern that, when exposed to a certain energy wavelength, emits characteristic colors and releases photons that the microscope system may collect and examine.

Immune system

Its capacity to provide highly contrasted, precise, and quantitative findings is what makes this approach so effective. The fluorescent labels’ brightness and contrast, as well as the antibodies’ specificity for their target antigens, determine the quality of the data. Because single or double target detection requires fewer instruments and requires less preparation, it has historically been used in the majority of labs. This method is constrained, nevertheless, especially when it comes to intricate research issues in fields like immune-oncology, where it’s critical to comprehend the interplay between many cell types, including T-cells, B-cells, and natural killer cells.

Display no less than five targets

These restrictions are lifted by multiplex immunofluorescence microscopy, which makes it possible to see up to five targets within cells or tissues at once. Icuradx provides clients with the option to build and verify unique panels in addition to pre-validated multiplex immunofluorescence panels, due to the complex optimization needed. Icuradx further enhances this with its high-content imaging, picture analysis, and thorough reporting. Without having to deal with the hassle of creating labor-intensive internal tissue processing, labeling, imaging, analysis, and reporting pipelines, customers can quickly turn their tissues into insightful information thanks to this integrated methodology.

A Strong Weapon

When it comes to immuno-oncology, where targeted treatments are critical, multiplex immunofluorescence is a powerful technique that may be used to find significant biomarkers and describe changes in their spatial distribution and activation states. As part of its ongoing commitment to progress cancer drug development, Icuradx provides pre-validated immune-oncology panels and is prepared to use these resources in order to meet your unique research needs. Speak with one of our staff members if you’d like additional information!

Advantages and drawbacks


may be used to collect multidimensional data on tissue architecture, the geographic distribution of different cell phenotypes, and the simultaneous coexpression of cell cycle and signaling markers in order to research cell biology.

Capable of evaluating many antibodies on a single tissue segment


need a much more time to gather, process, and evaluate.

requires more expensive machinery and specialized knowledge, both of which are currently lacking in qualified personnel