Immunofluorescence staining is one of the most effective methods in cell biology and pathology for looking at cells and tissues for antigens using immunohistochemistry using antibodies that have fluorescent proteins. Multicolour immunofluorescence staining in particular has democratised studies of layered biological structures, as multiple targets can be observed in the same specimen. The best cell biology and pathology way to look for cells and tissues expressing certain antigens through immunohistochemistry using antibodies expressing fluorescent proteins is immunofluorescence staining.

Multicolor Immunofluorescence Principles

Multicolour immunofluorescence – multiple colour fluorochrome-labelled antibodies for antigen labelling on an organ. Each fluorochrome is short wavelength when excited by shorter wavelength light, so you get lots of them all in one go with a fluorescence microscope and filters. It starts by sterilizing the sample (static, porous, locked) against shunt. On the sample are injected first-antibodies against the target antigens. Such primary antibodies are paired up with secondary antibodies using other fluorochromes. The fluorochromes you choose should be selected so that the emission spectra don't cross each other enough that the crosstalk between spectral signals can be reduced and staining optimized.

Figure 1. Multi-color immunofluorescence staining for pancreatic cell type determination. (Kalla D, et al.; 2021)

Applications of Multicolor Immunofluorescence Staining

1. Cancer Research and Diagnostics

Multicolour immunofluorescence images are used in cancer to define tumour cell identity and composition, to trace the microenvironment of the tumour, and to discover biomarkers of cancer onset and response to treatment. We could see tumor cells, immune cells and stromal cells in real time, for instance, to observe their behaviour and understand what causes tumours to grow and spread.

2. Neuroscience

Neuroscientists use multicolour immunofluorescence to examine nervous system circuitry. We can also see different neuronal and glial cell populations, synaptic proteins and signalling molecules within the same tissue slice. Such fine-grained mapping is needed to learn the cell- and molecular basis of neural development, function and pathology.

3. Immunology

The multicolour immunofluorescence of immunology is applied to narrow down the details of immune reactions. It's possible to look at how individual immune cell subpopulations within tissues co-operate and exchange, how cytokines and chemokines are secreted, how immune cells are turned on and moved. This kind of technology would be especially useful in studies of autoimmune disorders, infection and vaccine.

4. Stem Cell Research

Multicolour immunofluorescence staining is one of the most common methods used in stem cell studies to define stem cell differentiation capacity and to follow expression of pluripotency markers. Multiple differentiation markers can be identified simultaneously to learn about the diversity and lineage adherence of stem cells and to create regenerative treatments.

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Advancements in Multicolor Immunofluorescence Staining

The multicolor immunofluorescence staining technology has been modernized to add new capabilities and uses. Some of these advancements include:

1. Spectral Unmixing

Multicolour immunofluorescence is used in cancer to determine tumour cell identity and structure, to trace the tumour microenvironment, and to look for biomarkers of cancer progression and therapy response. For instance, we can see tumour cells, immune cells and stromal cells all at once to watch them, to see how they act, and identify what is motivating tumour growth and progression.

2. Advanced Imaging Systems

Multicolour immunofluorescence is now granular, with imaging instruments such as confocal and super-resolution microscopes enhancing sensitivity and resolution. These cameras produce high-definition images of subcellular organelles, as well as detection of low- abundance antigens, for cell and molecular analysis.

3. Multiplexing with Fluorescence Barcoding

When a fluorescence barcoded target is marked with several fluorochromes, the unique fluorescence signature of that target is generated. With this technique, many targets can be observed in a single sample at once, significantly increasing the multiplexing capability of immunofluorescence staining.

4. Automation and High-Throughput Screening

Multicolor immunofluorescence staining is now automated and accelerated by high-throughput screening technology, which can be performed on many large samples at very high reproducibility and accuracy. Automated staining equipment and image analysis software can measure fluorescence signals quickly and reliably and is accessible for scale-up experiments.

5. New Fluorochromes and Conjugation Chemistries

The appearance of new fluorochromes that were better photostable, brighter and spectrally sensitive increased the repertoire of fluorochromes for multicolor immunofluorescence staining. Furthermore, conjugation chemistries have made antibody labelling more efficient and accurate, as well as less noisy and thus better-quality.

Challenges and Future Directions

But even though multicolor immunofluorescence staining technology has been advancing quite beautifully, there are still challenges. Spectral overlap, photobleaching and non-specific binding can still be substantial limitations in getting good images. Furthermore, image interpretation for multicolour data demands image analysis tools and knowledge, thus further computational development and software is required.

In the future, the combination of multicolour immunofluorescence staining with other imaging techniques – electron microscopy, mass spectrometry – could reveal even more about cell and molecular function. It'll also be helped by the creation of new fluorescent probes and labelling techniques to further broaden the scope of applications.

Conclusion

Multicolor immunofluorescence staining has changed the way we look at living systems because many different targets can now be detected in a single sample. Applications in cancer, neuroscience, immunology and stem-cell biology opened new avenues of knowledge into the cell and molecular pathways of disease and wellbeing. This had been enhanced by imaging systems, spectral unmixing, multiplexing and automation. Until researchers discover and cure some of the old wounds, multicolour immunofluorescence will still undoubtedly be a core part of biomedical research, a source of discovery and progress.

1. Kalla D, et al.; The Missing Link: Cre Pigs for Cancer Research. Front Oncol. 2021, 11:755746.

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