Multiplex immunofluorescence: What is it?

Tyramide signal amplification and multispectral imaging form the basis of multiplex immunofluorescence (mIF), a high throughput immunofluorescence method that permits the simultaneous detection of several markers on a single tissue segment without damaging the tissue architecture.

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Fundamental characteristics

Using multispectral imaging, multiplex immunofluorescence creates pictures for analysis based on enzyme-based tyramide signal amplification.

After staining, all of the fluorophores on a single slide must be excited for multiplex immunofluorescence, but the emission spectral range cannot overlap with the excitation range.

The fundamental steps in analyzing the collected data include segmenting individual cells, segmenting the tissue into tumor and stroma, linearly unmixing the pictures, and phenotyping markers depending on markers of interest.

Multiple datasets detailing cell phenotypes and nearest neighbor distances may be created using subsequent data, and these datasets can be used to determine the geographical location of individual cells within a tissue.

Multiplex Immunofluorescence Microscopy: What is it?

With the use of fluorescent markers and various light wavelengths, multiplex immunofluorescence microscopy is a state-of-the-art technology that opens up a plethora of options for investigating various research questions. Since its founding, Icuradx has led the way in cell imaging, developing the first readily available tissue clearing reagents for multiplex immunofluorescent three-dimensional microscopy.

Illuminated Microscopy

When it comes to fluorescence microscopy, a dye-tagged antibody attaches itself to the target antigen to allow for the quantitative evaluation and viewing of certain areas of interest. When exposed to a certain energy wavelength, this target is represented as a fluorescent pattern that emits unique colors and releases photons that can be recorded and evaluated by the microscope equipment.


This method’s strength is its capacity to produce outcomes that are quantitative, precise, and strongly contrasted. The specificity of the antibodies for their target antigens and the contrast and brightness of the fluorescent labels determine the quality of the data. Due to its ease of planning and low equipment needs, single or double target detection has historically been the method used in the majority of labs. But this method has limitations, especially when it comes to intricate research issues in fields like immune-oncology, where it’s critical to comprehend how different cell types—including T-cells, B-cells, and natural killer cells—interact with one another.

Show up to five or more targets

These restrictions are lifted by multiplex immunofluorescence microscopy, which allows the simultaneous imaging of five or more targets inside of cells or tissues. Owing to the complex optimization required, Icuradx provides customers with the option to create and verify bespoke panels in addition to pre-validated multiplex immunofluorescence panels. Icuradx further enhances this with extensive reporting, image analysis, and high-content imaging. With the help of this integrated approach, customers can quickly convert their tissues into insightful information without having to worry about building labor-intensive internal pipelines for tissue processing, labeling, imaging, analysis, and reporting.

A Strong Instrument

Multiplex immunofluorescence is a powerful technique for discovering significant biomarkers and describing changes in their spatial distribution and activation states in the field of immuno-oncology, where targeted therapy are critical. Icuradx is dedicated to progressing the medication development process for cancer. They provide pre-validated immune-oncology panels and are prepared to use these resources to meet your unique research needs. Contact a member of our staff if you’d like further information!

Benefits and drawbacks


may be used to research cell biology by collecting multidimensional data on tissue architecture, the geographic distribution of various cell phenotypes, and the simultaneous coexpression of cell cycle and signaling markers.

able to evaluate many antibodies on a single piece of tissue


takes a great deal longer to gather, process, and evaluate.

expensive equipment and specialized knowledge, both of which are now lacking in qualified personnel

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