Development and Advantages of Multiplex Immunohistochemistry (mIHC)
Multiplex immunohistochemistry (mIHC) technology has advanced our understanding of complex samples such as tumors by revealing interactions between cells or cell populations, enabling deeper analysis of the tumor microenvironment. This approach provides significant momentum for clinical doctors, pathologists, and drug developers. Compared to traditional immunohistochemistry (IHC) and next-generation sequencing (NGS), mIHC offers precise spatial localization and multi-dimensional protein expression information overlaid in situ.
Huabio supports researchers in achieving more accurate and efficient mIHC experiments by enabling the simultaneous analysis of multiple immunological markers on a single FFPE slide. It can detect dozens of protein targets within tissue slices via fluorescence microscopy, facilitating high-resolution quantitative studies of the immune environment in both tumor and stromal tissues using minimal sample amounts.
Features and Applications of mIHC
● Multiplex immunohistochemistry (mIHC), evolved from standard single-target IHC, allows simultaneous detection of multiple targets on a single tissue section. It is a powerful tool for analyzing complex samples such as the tumor microenvironment, simultaneously obtaining information on tumor markers, cellular states, immune cell subtypes, immune regulation, and stromal cells, providing a comprehensive analysis of the tumor microenvironment.
● mIHC not only offers high-dimensional data similar to single-cell omics but importantly provides spatial context between various elements. It delivers detailed data essential for evaluating the dynamics of tumor-immune microenvironment interactions, playing an increasingly vital role in personalized medicine and companion diagnostics.
● TSA-based fluorescence amplification in mIHC facilitates detection across a wide range of protein abundances, even for low-expression proteins, making it ideal for protein colocalization studies.
● mIHC is a robust tool for researching complex disease biology, particularly when clinical samples are limited. Its capability to detect multiple markers in a single sample provides unprecedented insights into drug efficacy, toxicity, and resistance mechanisms, allowing comprehensive understanding of drug-tissue and drug-cell interactions. Consequently, this supports the development of more effective and safer therapeutics. Moreover, mIHC is useful for identifying prognostic and predictive cancer biomarkers, analyzing tumor-infiltrating lymphocytes, quantifying biomarkers, exploring upstream and downstream molecular relationships in signaling pathways, and molecular classification. Its compatibility with automated staining, whole-slide imaging, and validated image analysis workflows is critical for developing clinically relevant in vitro diagnostics.
Antibody Selection for Multiplex Experiments
Whether employing standard single-target IHC or mIHC, using high-quality and highly specific antibodies is critical. Huabio offers a selection of antibodies specifically validated for mIHC. Both novice researchers conducting mIHC experiments for the first time and experienced scientists looking to verify current protocols can trust Huabio's products and support services.
Antibodies suitable for mIHC exhibit excellent target specificity and stability due to rigorous initial screening through standard IHC and extensive mIHC validation. Huabio provides mIHC-validated antibodies for tumor-immune microenvironment markers, with 23 validated products currently available, as listed. Alongside providing reliable tools for tumor immunology research, Huabio also offers professional technical support and consultation services.
Example of multiplex IHC staining: Fluorescence multiplex immunohistochemical analysis of tertiary lymphoid structures in human small-cell lung carcinoma (FFPE). Figure A: Combined image with anti-CD20 (HA721138, green), anti-PD-L1 (HA721176, cyan), anti-CD56 (ET1702-43, magenta), anti-CD3 (HA720082, yellow), and DAPI (blue).
Staining protocol: HRP-conjugated UltraPolymer goat polyclonal antibodies HA1119/HA1120 as secondary antibodies. Stained using IRISKit™ (MH010101). Antigen retrieval: Heating at 95°C in Tris-EDTA buffer (pH 9.0) for 30 minutes. Counterstain: DAPI (blue).
Optimization and Validation Workflow for mIHC Detection by Huabio:
Optimize single-channel mIHC for each target. Identify optimal monoclonal antibody clones, antigen retrieval conditions, and antibody dilutions using positive control tissues.
mIHC panel composition. Test staining order based on target protein abundance and fluorescence signal intensity. Due to signal loss from repeated antigen retrieval, prioritize highly abundant targets for earlier staining and match low-abundance proteins with fluorophores exhibiting higher signal intensity.
Optimize staining conditions within multiplex protocols. Perform calibration experiments to ensure target-specific signals and balanced detection across the mIHC panel.
Compare mIHC panel staining with validated single-target IHC methods. Compare multiplex staining against corresponding validated single IHC staining on serial slides.
Perform precision testing. As the final stage, sequentially stain serial slides using target-specific antibodies or IgG isotype controls (negative controls).
