Multiplex Fluorescence Immunohistochemistry (mIHC): Consistent Performance Across Imaging Platforms
One Slide, Multiple Markers
Multiplex fluorescence immunohistochemistry (mIHC) detects multiple protein biomarkers on a single tissue section, making it possible to visualize cell phenotypes and their spatial interactions within the tumor microenvironment. Because every marker is resolved on the same slide, mIHC avoids the section-to-section misregistration and sample consumption that come with staining serial sections one marker at a time.
Principle: Tyramide Signal Amplification (TSA)
Our mIHC workflow is built on tyramide signal amplification (TSA), an enzymatic detection method that uses horseradish peroxidase (HRP) for high-density in-situ labeling of target biomolecules. Catalyzed by HRP and hydrogen peroxide, tyramide salts covalently bind to amino-acid residues at antigen–antibody sites and deposit biotin; cyclic amplification with streptavidin-HRP or fluorophores then dramatically boosts the detection signal.
- High-density in-situ labeling: signal is deposited directly at the antigen site, preserving spatial context.
- Broad dye compatibility: compatible with Alexa Fluor® dyes, standard fluorophores and colorimetric systems.
- Multiplex capability: sequential rounds of staining and signal inactivation resolve multiple markers on one section.
Verified on the Imaging Platforms You Already Use
A frequent question when setting up a multiplex panel is whether a kit will perform consistently on the laboratory's existing imaging system. To answer this, one identical staining protocol was applied to the same samples and imaged on the three platforms most commonly used in laboratories:
- 3DHISTECH Pannoramic MIDI III Digital Scanner
- Akoya Vectra Polaris
- ZEISS Digital Slide Scanner Axioscan 7
Uniform, high-quality results across all three systems confirm reliable kit performance independent of the imaging platform.
Figure 1. The same stained sections imaged on 3DHISTECH Pannoramic MIDI III, Akoya Vectra Polaris and ZEISS Axioscan 7.
Experimental Data
Experiment 1 — 3-plex panel
| Marker | Channel |
|---|---|
| Ki67 | 520 |
| PD-L1 | 620 |
| PANCK | 690 |
| MERGE | Composite |
Experiment 2 — 4-plex panel
| Marker | Channel |
|---|---|
| Ki67 | 520 |
| PD-L1 | 620 |
| PD-1 | 570 |
| PANCK | 690 |
| MERGE | Composite |
Tissue samples: tonsil and lung cancer. For each platform and each tissue type, single-marker images and the merged image are provided.
Figure 2. Tonsil and lung cancer sections stained with the 4-plex panel (Ki67-520, PD-L1-620, PD-1-570, PANCK-690) and the merged image.
Featured Products
Both kits are supplied as ready-to-use detection systems for TSA-based multiplex fluorescence IHC.
| Cat. No. | Product Name | Secondary Antibody |
|---|---|---|
| abs50015 | Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody) | Anti-mouse and rabbit |
| abs50031 | Absin 7-Color IHC Kit (Anti-Rabbit Secondary Antibody) | Anti-rabbit |
Request the Full Application Note
The complete Application Note includes the full imaging panel for both experiments across all three platforms. For the document or a quotation, please contact us at info@antbioinc.com.
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ANT BIO PTE. LTD. supplies high-quality reagents and solutions for life science research. Our sub-brands cover the full research workflow: Starter for antibodies and immunological assay kits, UA-Bio for recombinant proteins and drug discovery solutions, and Absin for general reagents and other detection kits. Explore our product portfolio at www.antbioinc.com.
