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Development of mIF

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With the advancement of immunotherapeutics, the urge to understand the complex tumor microenvironment has never been more pressing. Molecular histopathology advances allow for a shift from single-marker immunohistochemistry (IHC) to multiplexed marker detection.

Clinically, understanding of the tumor microenvironment, and subsequent selection of appropriately-targeted therapies, can be augmented by profiling and localizing immune checkpoint proteins. The applications of multiplex immunohistochemistry are numerous, and span clinical, translational, and basic research applications.

What is mIF?
Multiplex immunofluorescence (mIF) allows for the detection of multiple antigens on the same tissue section. It provides colocalization and spatial orientation of proteins, which facilitates an accurate determination of the target’s subcellular localization, identification of multiple cell types, and resolution of the relative proximity of biomarkers. More specifically, mIF assays provide information on the expression levels of biomarkers while increasing the number of biomarkers that can be visualized simultaneously on a single slide.

How Does it Work?
Simultaneous detection of multiple distinct proteins of interest within a single sample can be achieved with carefully optimized fluorescent mIF using tyramide signal amplification (TSA). This technique utilizes an unconjugated primary antibody specific to the protein of interest, and a primary specific secondary antibody conjugated to horseradish peroxidase (HRP). Detection is achieved with the fluorophore-conjugated HRP substrate, tyramide. When activated, tyramide forms covalent bonds with the tyrosine residues on or near the protein of interest. This permanent deposition allows the primary/secondary antibody pair to be stripped from the sample without disrupting the antigen-associated fluorescence signal. Thus, multiple rounds of staining can be performed in sequence on a given sample, without fear of the crosstalk that would otherwise result from using multiple primary antibodies raised in a single species.

The basic procedure for fluorescent mIF with TSA is as follows:

mIF staining cycle

Why mIF?
mIF using TSA provides a robust approach to tissue analysis enabling researchers to characterize molecular signaling or protein-protein interactions, understand the unique intricacies of the tumor microenvironment, and develop individually-tailored therapeutic interventions. mIF is a powerful technique for visualizing multiple targets of interest and provides several advantages over one-color or traditional multiplex IHC including:

  1. Maximize limited tissue samples
  2. Examine spatial arrangement of targets
  3. Wider dynamic and linear ranges
  4. Simplified panel design
  5. Clear counterstaining with DAPI
  6. Spectral un-mixing
  7. Streamlined quantitation

The Evolution and Future of mIF
A number of landmark developments have paved the way for immunohistochemistry since the 1940s when fluorescent labels were first conjugated to antibodies against targets of interest, including antibody purification and labeling, enzyme digestion and heat-induced epitope retrieval methods, signal amplification, and significant advancements to imaging technologies.

TSA and enhanced imaging capabilities have enabled the evolution of IHC beyond traditional or one-color approaches to mIF. Today, scientists can visualize a greater number of targets within a single tissue sample with even greater accuracy than ever before.

Moving forward, additional technical advancements and more widespread accessibility of imaging technology will likely see mIF transition to an integrated, workhorse clinical tool. Clinical use will require highly reproducible, quantifiable results, as well as adherence to regulatory requirements. However, such applications hold tremendous promise for making significant strides in clinical patient care, including characterization of the complex tumor microenvironment and subsequent selection of targeted therapies.



Below is the current listing of Bethyl antibodies involved in mIF research:


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