Multiplex IHC and Multiplex IF (mIHC and mIF) Service

Biological processes do not occur in isolation. In the complex landscape of tissue pathology—whether in the Tumor Microenvironment (TME), neurodegenerative plaques, or autoimmune lesions—the interplay between immune cells and stromal components is just as critical as their individual presence.

Multiplex Immunohistochemistry (mIHC) and Multiplex Immunofluorescence (mIF) move beyond the limitations of standard single-marker staining. By visualizing multiple targets simultaneously on a single tissue section, these technologies allow researchers to map spatial relationships, define complex cellular phenotypes, and preserve the critical morphological context of the tissue.

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How to choose
Workflow
Panels
Customized service

Choosing the Right Modality: mIHC vs. mIF

While both Multiplex Immunohistochemistry (mIHC) and Multiplex Immunofluorescence (mIF) aim to reveal the spatial biology of tissue, they utilize distinct detection systems suited for different investigative needs. Understanding the technical nuances and application scenarios of each is critical for study design.

Feature	Multiplex IHC (Chromogenic)	Multiplex IF (Fluorescent)
Detection Principle	Enzyme-mediated deposition of colored precipitates (e.g., DAB, Teal, Red).	Fluorophore excitation and emission (often via Tyramide Signal Amplification).
Imaging Hardware	Standard Brightfield Microscope / Scanner.	Fluorescence Microscope / Multispectral Scanner.
Plex Capability	Typically limited to 3–4 markers simultaneously to avoid color blending ambiguities.	High multiplexing potential (6+ markers) with narrow-band emission filters.
Co-localization	Difficult to quantify; colors mix physically on the slide (e.g., Brown + Red).	Excellent; signals are distinct channels that can be digitally overlaid and quantified.
Tissue Context	Superior visualization of tissue architecture and morphology without digital reconstruction.	Good, but relies on nuclear stains (DAPI) or autofluorescence for morphological context.
Data Permanence	Permanent; slides do not fade and can be archived for years.	Signal can fade (photobleaching) over time; digital archiving is essential.

Workflow of mIHC and mIF

High-order multiplexing requires a shift from traditional "cocktail" staining to a sequential, iterative approach. Whether utilizing the morphological clarity of mIHC or the high-plex capabilities of mIF, our workflow allows for the use of multiple primary antibodies from the same host species without cross-reactivity.

Our optimized protocol follows a cyclic "Stain-Image/Strip-Repeat" logic (for digital mIHC) or a "Stain-Strip-Stain" logic (for mIF), ensuring epitope stability and signal independence.

Phase 1: Foundation (Common to mIHC & mIF)

Deparaffinization & Rehydration: Standard tissue preparation.
Antigen Retrieval (HIER): A single, robust heating step unmasks epitopes. Our buffers are formulated to protect tissue morphology even through subsequent heating cycles.
Blocking: Application of a universal blocking buffer to neutralize endogenous enzymes and prevent non-specific binding.

Phase 2: The Staining Cycle (Repeated for each Target)

Primary Antibody Incubation: Binding of the specific primary antibody to the target antigen.
HRP-Secondary Introduction: A polymer-HRP secondary antibody binds to the primary.
The Divergence Point (Detection):
- For mIF: A Tyramide-Fluorophore is added. The HRP catalyzes the covalent deposition of the fluorophore directly onto the tissue.
- For mIHC: A Tyramide-Chromogen or high-sensitivity substrate (e.g., DAB, Teal, Red) is added to create a stable, colored precipitate.
Antibody Stripping / Heat Deactivation: The slide is heated. This strips away the primary and secondary antibody complex, but the covalently bound fluorophore remains.

Phase 3: Counterstaining & Imaging

Counterstaining:

mIF: DAPI is used to visualize nuclei and assist in cell segmentation.
mIHC: Hematoxylin provides the nuclear contrast and anatomical context.

Imaging:

mIF: Multispectral scanning or fluorescence microscopy captures distinct spectral channels.
mIHC: Standard brightfield scanning captures the composite colored image.

mIHC&mIF workflow

Featured Application Panels

Our multiplex panels are curated to provide deep insights into specific biological mechanisms. By combining structural markers with functional and lineage-specific markers, these panels provide a holistic view of the cellular landscape.

Multiplex IF (Human Validated Panels) (mIFH Panels for human FFPE tissue specimens)

Panel Name	Target Markers	Primary Application
Checkpoint Panel 1	PD-L1, CD8, CD56, IFNγ, SOX10/PanCK, DAPI	Tumor immunity & NK cell profiling
Checkpoint Panel 2	GAL9, LAG3, CD8, TIM3, CD155, MHCII	Exhaustion & inhibitory receptor mapping
T-Cell Activation	CD3, CD4, CD8, CD45RO, Grzm B, ICOS1, Ki67, DAPI	Activation state & memory profiling
T-Helper Lineage	CD3/CD4, CD8, FoxP3, T-bet, GATA3, RORγ, DAPI	Th1/Th2/Th17/Treg differentiation
Plasma Cell	CD3/CD4, CD8, CD20, IgG, CD138, Ki67, DAPI	B-cell maturation & antibody secretion
Myeloid Panel	CD11b, CD1a, GAL9, CD68, CD83, CD40, DAPI	Macrophage/DC polarization & activation
TLS Panel 1	CD3/CD4, CD8, CD20, PNAd, DC-Lamp, Ki67	Tertiary Lymphoid Structure (TLS) architecture
TLS Panel 2	MADCAM, CD8, CD20, PNAd, RORγ, OLIG2, DAPI	TLS formation & neogenesis
IL-10 Panel	CD3/CD4, CD8, CD20, CD34, T-bet, IL-10, DAPI	Anti-inflammatory cytokine mapping
Immune Suppression	CD34, IDO1, ARG1, CD39, CD73, DAPI	Metabolic suppression & MDSC profiling
Ab Production	CD3/CD4, CD8, CD20, IFNγ, IgG, Ki67, DAPI	Functional B-cell analysis

Multiplex IF (Mouse (Murine) Validated Panels) (mIFH Panels for murine FFPE tissue specimens)

Panel Name	Target Markers	Primary Application
Murine TLS	CD4, CD8, CD19, CD11c, PNAd, CD34, DAPI	Characterizing lymphoid structures in mouse models
Murine T-Cell Activity	CD4, CD8, CD19, Grzm B, CD34, Ki67, DAPI	Cytotoxic activity & proliferation in mice

Multiplex IHC Panels

Panel Name	Target Markers	Primary Application
T-Cell Activation	CD3, Granzyme B, Ki67, PanCK	Cytotoxic T-cells in tumor nests
PD-L1 Checkpoint	CD8, CD68, PD-L1, PanCK	PD-L1 status on tumor vs. macrophages
Memory T-Cell	CD3, CD45RO, PD-1, PanCK	Exhausted memory cells in TME
APC Panel	CD11b, CD68, CD20, MHC II	Antigen presentation landscape

Custom Panel Design Services

Every research hypothesis is unique. While our pre-validated panels cover core applications, novel discoveries often require unique marker combinations.

Developing a multiplex panel is complex; it requires balancing antibody species, fluorophore spectral overlap, and the order of antigen retrieval. Our Custom Panel Design service handles this complexity for you.

Our Service Includes:

Consultation: Feasibility assessment of your target markers.
Validation: Rigorous testing of antibody specificity and sensitivity.
Optimization: Balancing signal-to-noise ratios for every channel.
Protocol Transfer: We provide a fully optimized protocol ready for your lab.

* For Research Use Only. Not for use in diagnostic procedures.

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From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

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Dr. Sarah Mitchell, Research Scientist

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