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Multiplex IHC: Visualising Multiple Antigens in a Single Tissue Section
- Eghosa Arovo
- 12 hours ago
- 4 min read
See more from every slide: how multiplex Immunihistochemistry (IHC) lets you detect multiple proteins in one tissue section.
Introduction
Last week, we explored how moving beyond brown DAB and into coloured chromogens can add a new layer of insight to your histology research. By using red, purple, green, yellow, teal, and other chromogens, researchers can highlight different proteins with far better contrast and clarity than single-colour IHC alone.
In this blog, we take the next step: multiplex immunohistochemistry (mIHC).
Multiplex IHC allows you to detect multiple antigens on the same tissue section, with each antibody visualised using a different chromogen. This approach dramatically increases the informational bandwidth of a single slide—saving tissue, time, and cost, while delivering far richer biological context.
What is multiplex chromogenic IHC?
In standard single-plex IHC, one antibody is used to detect one protein target, usually visualised with a brown DAB chromogen. If you want to study three markers, you typically need three serial sections.
In multiplex chromogenic IHC, multiple primary antibodies are applied sequentially to the same tissue section. Each antibody is detected using a distinct enzyme–chromogen system, producing a different colour for each antigen.(abcam.com)
At LabNexus, we support multiplex IHC using a wide chromogen palette:
Red, Purple, Teal, Blue, Green, Yellow, DAB (brown), and Silver
This allows researchers to design highly flexible panels tailored to their biological question—whether that’s immune profiling, tumour–stroma interaction, or co-expression of signalling markers.
Why multiplex instead of single-plex IHC?
Multiplex IHC offers several major advantages for tissue-based research:
1. More data from limited tissueClinical and preclinical samples are often scarce. Multiplexing reduces the number of sections needed, preserving valuable tissue while extracting more information per slide.
2. True spatial contextBecause all markers are on the same section, you can see exactly how different proteins are arranged relative to one another—something that serial sections can only approximate.
3. Stronger biological interpretationMultiplexing allows you to answer more complex questions, such as:
Which immune cells are physically interacting with tumour cells?
Which stromal cells express activation markers?
Which proliferating cells also express a therapeutic target?
Case example: PanCytokeratin (Yellow) + CD8 (Silver)
To show how multiplex chromogenic IHC increases the amount of information you can extract from a single tissue section, we stained human colon control tissue with two well-known markers: Pan-cytokeratin (PanCK) and CD8.
First, PanCK was stained alone using a yellow chromogen, clearly highlighting the epithelial cells lining the colonic glands:
Next, CD8 was stained alone on a separate section using a silver chromogen, marking cytotoxic T cells scattered throughout the tissue:
Finally, both antibodies were applied together on a third colon control section using multiplex IHC:
In this dual-stained section, epithelial cells appear yellow (PanCK), while CD8-positive immune cells appear silver. Where CD8-positive lymphocytes are closely associated with PanCK-positive epithelium, the two colours visually overlap, producing mixed colour regions.
This demonstrates the classic “1 + 1 = 3” effect:
Yellow = epithelial cells (PanCK)
Silver = cytotoxic T cells (CD8)
Mixed yellow–silver tones = immune cells interacting with epithelium
Rather than just showing that both markers are present, multiplex IHC reveals how they are positioned relative to each other—a key advantage for studies of immune infiltration, epithelial–immune interactions, and tumour immunology.
Designing a good multiplex IHC panel
Effective multiplex IHC requires thoughtful panel design. Key considerations include:
Target abundance: High-expression markers can dominate visually. These are often paired with lighter chromogens or weaker enzyme systems.
Subcellular location: Nuclear, cytoplasmic, and membranous markers are easier to separate visually when multiplexed.
Chromogen contrast: Colour choices must be distinct enough to avoid confusion. For example:
Yellow + red + teal = excellent separation
Brown + purple + silver = strong contrast for dense tissues
Antibody compatibilityAntibodies must be from different species or used with sequential stripping methods to prevent cross-reactivity.(abcam.com)
At LabNexus, we assist researchers with panel design, chromogen selection, and protocol optimisation to ensure clean, interpretable results. Book your Free Consultation with us today if you plan on doing a panel.
Elevate your histology research with multiplex at LabNexus
At LabNexus, we specialise in research-only histology services for cancer and tissue researchers across London and the UK. We offer:
Single-plex and multiplex chromogenic IHC
A full chromogen palette:Red, Purple, Teal, Blue, Green, Yellow, DAB, and Silver
High-quality tissue processing, embedding, and sectioning
Slide scanning for digital and quantitative analysis
Optional TMA construction for high-throughput studies
All multiplex staining is performed using our state-of-the-art Ventana Ultra Benchmark platforms, ensuring high reproducibility, excellent signal-to-noise ratios, and consistent turnaround times.
We deliver fast turnaround, affordable pricing, and expert scientific support—without the need for you to run your own histology facility.
Please note: LabNexus provides histology services for research purposes only. We do not process diagnostic samples.
Conclusion
Multiplex chromogenic IHC transforms histology from a one-marker-at-a-time technique into a multi-dimensional tissue analysis platform. By detecting several antigens on a single section, and using colour mixing to reveal co-localisation, researchers can:
Extract more data from limited tissue
Preserve spatial relationships between biomarkers
Generate clearer, more biologically meaningful results
Whether you want classic DAB IHC, bright single-colour stains, or advanced multiplex panels, LabNexus can support your research with high-standard histology services.
If you’re ready to elevate your tissue research with multiplex staining, we’d be happy to help.
References
Slide images are from samples processed, embedded, cut and stained by LabNexus and partner laboratories.
Abcam. Multiplex immunohistochemistry (mIHC): technical overview.https://www.abcam.com/en-us/knowledge-center/immunohistochemistry/multiplex-immunohistochemistry-mihc
Leica Biosystems. Tips & Tricks to Multiplexing: Choosing chromogen colours.https://www.leicabiosystems.com/articles/ls-articles/tips-tricks-to-multiplexing-how-to-choose-chromogen-colors-for-multiplex-and/
Ramos-Vara, J.A. (2005). Technical aspects of immunohistochemistry. Veterinary Pathology, 42(4), 405–426.
Maiques, O. et al. (2022). Multiplex chromogenic immunohistochemistry to stain and analyse two markers in paraffin tissue sections. MethodsX, 9, 101788.
Bancroft, J.D., & Gamble, M. (2020). Theory and Practice of Histological Techniques (8th ed.). Elsevier.
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