Histology 101: What Is Immunofluorescence (IF)?

New to immunofluorescence? Here’s a simple guide to what IF is and why it’s used in tissue research.

IF for Phalloidin and DAPI in Human Bone Marrow-derived Mesenchymal Stem Cells - STAINSFILE

Introduction

As histology research moves beyond single-marker stains and into more complex, multi-target analysis, immunofluorescence (IF) has become an increasingly important technique.

If you’ve worked with immunohistochemistry (IHC) before, IF follows the same basic principle—using antibodies to detect specific proteins in tissue. The key difference is how the signal is visualised.

In this Histology 101 blog, we explain what immunofluorescence is, how it works, and why researchers use it in cancer and tissue studies.

What is immunofluorescence (IF)?

Immunofluorescence (IF) is a staining technique that uses fluorescent dyes (fluorophores) attached to antibodies to detect and visualise specific antigens in cells or tissue sections.

Instead of producing a coloured precipitate like chromogenic IHC (e.g. brown DAB or red chromogen), IF produces a fluorescent signal that glows when excited by a specific wavelength of light under a fluorescence microscope.

In simple terms:

• IHC = colour you can see in brightfield microscopy

• IF = light you can see only under fluorescence microscopy

The brighter the fluorescence, the more antigen is present at that location in the tissue.

How does IF work?

The Principle of Immunofluorescence Assays - Ibidi

The basic workflow of IF is very similar to IHC:

1. The tissue is fixed, processed, embedded, and sectioned.

2. A primary antibody binds to the target protein (antigen).

3. A fluorescently labelled secondary antibody binds to the primary antibody(or a directly labelled primary antibody can be used).

4. The tissue is counterstained (often with DAPI to label nuclei).

5. The slide is visualised using a fluorescence microscope or scanner.

Each fluorophore emits light at a specific wavelength, allowing multiple targets to be detected using different colours (e.g. green, red, far-red, etc.).(Abcam, 2023)

What is IF used for in tissue research?

Immunofluorescence is widely used in:

• Cancer research – mapping tumour markers, immune infiltration, and signalling pathways

• Cell biology – tracking protein localisation inside cells

• Developmental biology – studying differentiation and tissue organisation

• Neuroscience – identifying neuronal subtypes and synaptic markers

• Stem cell research – confirming lineage markers and differentiation states

Because IF allows multiple markers to be visualised at once, it is especially valuable for studying co-expression, cell identity, and protein localisation within complex tissues.

IF vs chromogenic IHC: what’s the difference?

Both IF and IHC detect proteins using antibodies, but they differ in how signals are produced and analysed.

Chromogenic IHC

• Produces coloured signals (e.g. brown DAB, red chromogen)

• Viewed under a standard brightfield microscope

• Slides are permanent and easy to archive

• Excellent for routine staining and pathology-style analysis

  
  

Immunofluorescence (IF)

• Produces fluorescent signals

• Requires a fluorescence microscope or scanner

• Allows high-level multiplexing (many markers at once)

• Highly sensitive and quantitative

• Signals can fade over time (photobleaching)

In practice, IF is often used when high sensitivity or high multiplexing is needed, while chromogenic IHC is preferred for robust, archive-ready slides.

Why researchers use IF

Researchers choose immunofluorescence when they need to:

• Detect low-abundance proteins

• Visualise multiple markers in one tissue section

• Study co-localisation and protein interactions

• Perform quantitative image analysis

• Integrate tissue staining with AI or spatial biology workflows

IF is especially powerful for mapping complex cellular environments like the tumour microenvironment or immune niches in tissue.(Fridman et al., 2012)

Single-plex vs multiplex IF

Just like IHC, IF can be:

• Single-plex – one antibody, one fluorophore

• Multiplex – several antibodies, each with a different fluorophore

Multiplex IF allows researchers to detect 4–10+ markers on the same tissue section (and even more with cyclic workflows), making it a cornerstone of modern spatial biology.(Abcam, 2023)

Conclusion

Immunofluorescence is a powerful extension of traditional histology that allows researchers to visualise proteins with high sensitivity and multiplexing capability.

By using fluorescently labelled antibodies, IF makes it possible to:

• Detect multiple antigens in a single tissue section

• Study co-localisation and spatial relationships

• Generate quantitative, high-resolution tissue data

Whether you are just starting with IF or planning a multiplex spatial biology project, LabNexus can support your research with high-quality IF staining and imaging services.

Book a free consultation:https://www.labnexus.co.uk/book-consultation

References

1. IF Image from StainsFile: https://www.stainsfile.com/theory/methods/immunostaining-immunofluorescence/

2. The Principle of Immunofluorescence Assays (Diagram): https://ibidi.com/content/364-the-principle-of-immunofluorescence-assays

3. Abcam. Immunofluorescence (IF) staining overview.https://www.abcam.com/en-us/knowledge-center/immunofluorescence/if-staining

4. Coons, A.H., Creech, H.J., & Jones, R.N. (1941). Immunological properties of an antibody containing a fluorescent group. Proceedings of the Society for Experimental Biology and Medicine, 47, 200–202.

5. Fridman, W.H. et al. (2012). The immune contexture in human tumours: impact on clinical outcome. Nature Reviews Cancer, 12, 298–306.

6. Pendergraft, S.S., & Preston, G.A. (2015). Methods in immunofluorescence. Methods in Molecular Biology, 1274, 3–16.