Introduction

Multicolor IHC staining has remade histopathology with multiple biomarkers showing up in the same tissue slice. It is a powerful instrument that enables scientists and clinicians to have a great deal of understanding about where and how cells behave in space, and then use that knowledge to do even more sophisticated diagnostics and studies in many different conditions, particularly cancer. Multicolor IHC staining with individual chromogenic substrates or fluorescent tags allows us to visualize microenvironments, cell details and molecular dynamics of each tissue up-close.

Figure 1. Histochemical and immunohistochemical staining of the femur fracture sample and comparison between micro-CT and multicolor staining images (decalcified with EDTA). (Sun Y, et al.; 2022)

Principles of Multicolor IHC Staining

The concept of antibodies recognizing living-tissue specific antigens in a living tissue was then translated to immunohistochemistry, to find specific antigens in tissue sections. Multicolor IHC staining does this an extra step by staining multiple antibodies with a different label to capture multiple targets simultaneously.

1. Main Antibody Binding: It starts from the main antibodies that are native for the antigens of the tissue.
2. Secondary Antibody Sticking: Secondary antibodies attached to enzymes or fluorophores attach to primary antibodies. These second antibodies are individual, and up-regulate the signal.
3. Signals: Depending on the label used, signals are read chromogroscopically or fluorescently. In chromogenic detection, enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which undergo a reaction, make the colour. Fluorescent detecting uses excited fluorophores.

Chromogenic Multicolor IHC Staining

There are enzyme substrates in chromogenic staining, that are colored by enzyme reactions. And there are chromogens: 3,3'-diaminobenzidine (DAB), brown; 3-amino-9-ethylcarbazole (AEC), red. Multicolor chromogenic IHC is often done in serial staining each antigen stain separately with different chromogen.

1. Sequential Staining: Primary antibodies, secondary antibodies are sequentially stain along with a chromogen development of a different chromogen is used to identify multiple targets.
2. Avoid Cross-Reactivity: To avoid cross-reactivity, between staining sessions antibody stripping or blocking is performed.
3. Compatibility with colours: Choose compatible chromogens to avoid signal noise.

Fluorescent Multicolor IHC Staining

Fluorescent IHC staining relies on fluorophores that emit light at certain wavelengths when excited. It's worth trying out: you get ultra-sensitive, you can multiplex targets, and it's digitally integrated into your scanner.

1. Direct and Indirect: Direct Labelling: Primary antibodies are paired with fluorophores and indirect labelling using secondary antibodies paired with fluorophores.
2. Spectral Overlap Reduction: Avoid the fluorophores which are the spectrally least overlapped fluorophores or you'll end up with signal bleeding through and false interpretation.
3. Multispectral Imaging: High-tech imaging devices with spectral unmixing allow overlap signals to be distinguished from one another and multicolor fluorescent IHC is stronger.

Applications of Multicolor IHC Staining

1. Cancer Research and Diagnostics: Multicolor IHC is used widely in oncology to study a tumor microenvironment, tumor-infiltrating lymphocytes and multiple biomarker expression simultaneously. It's how we determine tumor heterogeneity, immune escape and therapy.
2. Neuroscience: In neuroscience, multicolor IHC makes neural circuitry, neurotransmitter systems and neurodegenerative disease pathology visible by showcasing many markers in one piece of tissue.
3. Stem Cell Research: This tool comes in handy for stem cell research because we can track differentiation tracks and identify the microenvironments that regulate stem cell behavior.
4. Diseases Related To Infections:Multicolor IHC can be used to investigate the host-pathogen interaction, to depict the spread of pathogens in tissues, and to monitor immune responses.

Advantages and Challenges

Advantages:

1. Tissue Map Complete: Multiple Target detection gives you the complete view of tissues structure and molecular interaction in one go.
2. Accurate Diagnostic Match: Multicolor IHC is more precise because multiple biomarkers are paired to the same tissue.
3. Time and Money Saver: Multi stain assay means smaller tissue sections, single staining steps and saves time and money.

Challenges:

1. Antibody Optimization: The primary and secondary antibodies must be optimized to be specific and cross-reactive.
2. Signal Interference: A high incidence of signal interference, in the form of spectral mixing in fluorescent IHC and mixing of colors in chromogenic IHC (labels must be carefully selected and validated).
3. Technical Complexity: The multicolor IHC protocols can be technical, involving exact tuning of the experimental conditions and a large amount of validation.

Future Perspectives

Multicolour IHC staining is constantly evolving to find more and better diagnostic and biological information. Automated staining infrastructure, fluorophore chemistry and digital pathology/artificial intelligence will be the next steps to bringing multicolor IHC even closer to accuracy, reproducibility and accessibility.

1. Digital Pathology: Interfaces with high-resolution digital imaging and automated image analysis software for quantitative interpretation and independent interpretation of multicolor IHC images.
2. Artificial Intelligence: AI algorithms are developed to analyse complex staining patterns, diagnose diseases and make diagnosis decisions.
3. Nanotechnology: Nanoparticles as tags in IHC could be used to boost signal strength and multiplexing.

Conclusion

Multicolour immunohistochemical staining technologies sit on the vanguard of contemporary pathology and biomedical research and open up a whole new world of data about tissue biology and disease progression. Its ability to ingest more than one biomarker from the same tissue slice helps not only in diagnosing but in understanding complex biological systems. The technology is always getting better and better, but multicolor IHC will be a new and increasingly valuable tool in the laboratory and at the bedside for diagnosing, treating and individualized medicine.

1. Sun Y, et al.; Multicolor Histochemical Staining for Identification of Mineralized and Non-Mineralized Musculoskeletal Tissue: Immunohistochemical and Radiological Validation in Decalcified Bone Samples. Bioengineering (Basel). 2022, 9(10):488.

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