mIHC Multiplex Fluorescent Staining Kits
mIHC Multiplex Fluorescent Staining Kits
Multiplex immunohistochemistry / multiplex immunofluorescence (multiplex IHC / multiplex IF, abbreviated as mIHC) is built on the Tyramide Signal Amplification (TSA) system, enabling highly sensitive spatial co-localization detection of multiple protein targets on the same tissue section. It is particularly suitable for tumor immune microenvironment assessment, studies of immunotherapy response and resistance mechanisms, as well as biomarker screening in the preparatory stage of spatial omics.TSA utilizes HRP to catalyze the covalent deposition of tyramide-conjugated fluorophores around the target protein, significantly amplifying the signal while preserving good tissue morphology and spatial information. Combined with multiple rounds of antibody stripping and tyramide substrates of different emission wavelengths, a 3–9 color multiplex labeling system can be established. Based on TSA-mIHC technology, Aladdin’s 3–9 color multiplex fluorescent staining kits have been systematically optimized for FFPE and frozen tissue sections, providing standardized tools for routine pathology laboratories to rapidly build multiplex fluorescence platforms.
I. Overview of mIHC / Multiplex Fluorescent Staining Technology
The core objective of multiplex immunofluorescence is to simultaneously resolve cell types, functional states, and spatial relationships on the same tissue section. Using the tumor immune microenvironment as an example, researchers often wish to observe, within a single FFPE section, tumor cell markers, T cell subsets, B cells, myeloid cells, as well as functional molecules such as PD-1/PD-L1 and Ki-67 at the same time. If only single-color DAB or 1–2 color conventional immunofluorescence is used, multiple serial sections are required, which not only consumes precious samples but is also affected by inter-section heterogeneity, making accurate spatial statistics difficult.
TSA-mIHC achieves sequential multi-round staining and covalent deposition of fluorescent signals, overlaying multiple labeling channels on one single section. This approach combines high spatial resolution with a certain degree of quantitative capability, allowing multi-parameter joint evaluation of cell populations of interest directly on the same slide.
II. Principle of TSA Tyramide Signal Amplification
In TSA-mIHC, a typical staining round can be summarized as follows:
1) The primary antibody recognizes the target antigen and localizes to the cell membrane, cytoplasm, or nucleus.
2) An HRP-conjugated secondary antibody binds to the primary antibody, thereby localizing HRP around the target.
3) A working solution containing fluorescent tyramide substrate is added; in the presence of H₂O₂, HRP catalyzes the formation of highly reactive free radicals.
4) Activated tyramide covalently binds to nearby proteins (mostly tyrosine side chains), generating high-density fluorescent deposits.
5) Specific stripping buffer or heat-induced conditions are used to remove the primary antibody and HRP-conjugated secondary antibody, while preserving the deposited fluorescent signal.
6) A new primary antibody and a tyramide substrate of a different wavelength are then applied, entering the next staining round.
By repeating this cycle, 3–9 different fluorescent signals with distinct wavelengths can be sequentially accumulated on the same section to achieve multiplex co-localization. The key advantage of TSA is that the signal originates from covalent deposition, providing high sensitivity and high stability; the signal is not easily removed during subsequent stripping and processing steps.
III. Advantages of TSA-mIHC
Compared with conventional immunofluorescence, TSA-based mIHC has the following features:
1) High sensitivity: Suitable for detecting low-abundance targets, especially beneficial for weak-signal molecules such as PD-L1 and certain phosphorylated proteins.
2) Intact spatial information: Expression intensity, subcellular localization, and neighborhood relationships can be obtained simultaneously at the single-cell level.
3) Sample saving: Multiple targets are detected on a single section, making it suitable for scenarios with limited sample amount, such as fine-needle biopsies or small animal tissues.
4) Good compatibility: Applicable to routine FFPE tissues, frozen sections, or cell slides, allowing easy integration into existing pathology workflows.
5) Ready for digital pathology: Multichannel fluorescent images can be imported into image analysis and spatial statistics software, improving the objectivity of data interpretation.
IV. Typical Application Scenarios
1) Tumor Immune Microenvironment Analysis
On the same tumor tissue section, simultaneous labeling of tumor cells, T cells, B cells, macrophages/dendritic cells, and immune checkpoint molecules enables acquisition of three-dimensional information linking “cell type–functional marker–spatial location.” This supports immune microenvironment profiling and therapeutic response prediction.
2) Pathological Subtyping and Diagnostic Research
For diseases that share similar morphology but differ in molecular mechanisms (e.g., lymphoma subtypes, lung cancers with different driver mutations), mIHC can simultaneously evaluate multiple diagnostic/prognostic markers on limited pathology samples, thereby improving tissue utilization efficiency.
3) Signaling Pathway and Co-localization Studies
By labeling total protein and phosphorylated protein, receptor and ligand, transcription factor and downstream targets within the same cell or tissue region, TSA-mIHC enables fine-grained analysis of spatial co-localization and pathway activation states.
4)Drug Mechanism of Action and Companion Research
Before and after drug treatment, multiplex fluorescent detection of multiple markers within the same pathway helps dissect drug action steps, feedback regulation, and resistance-related changes, supporting candidate biomarker screening and companion diagnostic development.
V. Design of Multicolor Panels and Layout of Aladdin 3–9 Color Multiplex Fluorescent Staining Kits
1. Selection of Channel Number
Aladdin multiplex fluorescent staining kits cover different channel numbers from three to nine colors, allowing users to design stepwise according to project complexity:
1) Three-color and four-color panels are suitable as entry-level schemes, used for co-localization validation, method establishment, and simple “cell type + functional marker + nuclear stain” combinations.
2) Five-color and six-color panels are appropriate for most routine immune microenvironment analyses, such as simultaneously labeling tumor cells, multiple immune cell types, and 1–2 functional molecules.
3) Seven-color and above panels are suitable for integrating more immune subsets and pathway markers, for complex microenvironment dissection or high-dimensional panel development.
Experimentally, 3–4 color kits can first be used to optimize antigen retrieval conditions, primary antibody dilutions, and staining order. Once parameters are stabilized, the system can then be gradually upgraded to 6–9 colors.
2. Selection Between Anti-Rabbit Secondary and Rabbit/Mouse Universal Secondary Systems
This kit series is divided into “anti-rabbit secondary antibody type” and “rabbit/mouse universal secondary antibody type” according to the secondary antibody system, corresponding to different primary antibody combination strategies:
1) When all or the vast majority of primary antibodies in the panel are rabbit-derived, the anti-rabbit secondary antibody type can be prioritized. This configuration is simpler and easier to control background.
2) When the panel includes both rabbit and mouse primary antibodies, or is expected to be expanded to mixed-species antibodies in the future, it is recommended to directly choose the rabbit/mouse universal secondary antibody type, in order to reduce the workload associated with later system switching.
3. Product Portfolio and Application Positioning
Catalog No. | Product Name | Recommended Application Summary |
3-Color Multiple Fluorescent Staining Kit (Anti-Rabbit Secondary Antibody) | Suitable for basic three-color panels using only rabbit primary antibodies, such as tumor marker + single immune cell marker + nuclear staining. | |
3-Color Multiple Fluorescent Staining Kit (Anti-Rabbit and Mouse Secondary Antibody) | Suitable for three-color schemes combining rabbit and mouse primary antibodies, for method development or simple co-localization analysis. | |
4-Color Multiple Fluorescent Staining Kit (Anti-Rabbit Secondary Antibody) | Suitable for four-color panels mainly based on rabbit primaries, for example tumor marker + two immune cell lineages + functional molecule. | |
4-Color Multiple Fluorescent Staining Kit (Anti-Rabbit and Mouse Secondary Antibody) | Suitable for four-color combinations requiring simultaneous detection of rabbit and mouse primary antibodies, and convenient later expansion to higher-plex panels. | |
5-Color Multiple Fluorescent Staining Kit (Anti-Rabbit Secondary Antibody) | Designed for moderately complex rabbit-only panels, allowing simultaneous detection of multiple cell types plus 1–2 functional markers. | |
5-Color Multiple Fluorescent Staining Kit (Anti-Rabbit and Mouse Secondary Antibody) | Suitable for routine tumor immune microenvironment panels using both rabbit and mouse primary antibodies on the same section. | |
6-Color Multiple Fluorescent Staining Kit (Anti-Rabbit and Mouse Secondary Antibody) | For more complex immune microenvironment analysis, covering multiple immune subsets and key functional molecules. | |
6-Color Multiple Fluorescent Staining Kit (Anti-Rabbit Secondary Antibody) | Ideal for laboratories with established rabbit monoclonal systems to upgrade from single-plex to six-plex spatial staining. | |
7-Color Multiple Fluorescent Staining Kit (Anti-Rabbit Secondary Antibody) | For high-dimensional rabbit-only panels integrating multiple pathways and cell subsets on the same section. | |
7-Color Multiple Fluorescent Staining Kit (Anti-Rabbit and Mouse Secondary Antibody) | Suitable when complex panels require simultaneous use of multiple rabbit and mouse primary antibodies. | |
Eight Color Multiplex Immunofluorescence Kit Plus Version (Anti-Rabbit Secondary Antibody) | Supports high-dimensional rabbit-only labeling schemes, suitable for in-depth analysis of the tumor immune microenvironment or other complex tissues. | |
8-Color Multiple Fluorescent Staining Kit (Anti-Rabbit and Mouse Secondary Antibody) | For complex panel development, enabling integration of existing rabbit and mouse antibody resources within one system. | |
9-Color Multiple Fluorescent Staining Kit (Anti-Rabbit Secondary Antibody) | Suitable for the highest-plex rabbit-only panels, enabling spatial co-localization of nine markers on a single section. | |
9-Color Multiple Fluorescent Staining Kit (Anti-Rabbit and Mouse Secondary Antibody) | Designed for ultra-high complexity panels, supporting joint analysis of multiple cell types and functional pathways in the same tissue. |
The tyramide fluorophores used in our kits include one or more of the following: TYR-480, TYR-520, TYR-570, TYR-620, TYR-700, TYR-780, TYR-650, TYR-540, and TYR-690.
Dye | Excitation wavelength (nm) | Emission wavelength (nm) |
DAPI (blue) | 350 | 420 |
TYR-480 fluorescent dye (concentrate, 200×) | 450 | 480 |
TYR-520 fluorescent dye (concentrate, 200×) | 490 | 520 |
TYR-570 fluorescent dye (concentrate, 200×) | 550 | 570 |
TYR-620 fluorescent dye (concentrate, 200×) | 590 | 620 |
TYR-690 fluorescent dye (concentrate, 200×) | 640 | 690 |
TYR-780 fluorescent dye (concentrate, 200×) | 750 | 780 |
TYR-700 fluorescent dye (concentrate, 200×) | 680 | 700 |
TYR-650 fluorescent dye (concentrate, 200×) | 630 | 650 |
TYR-540 fluorescent dye (concentrate, 200×) | 515 | 540 |
VI. Frequently Asked Questions and Troubleshooting
1) High background signal
When background signal is elevated, check whether peroxidase blocking is sufficient, whether the blocking buffer is appropriate, and whether the primary antibody dilution is too low. If necessary, increase the washing strength or add a tissue autofluorescence quenching step.
2) Signal attenuation in later staining rounds
When signals in the later rounds are markedly weaker, evaluate whether the antibody stripping or repeated retrieval conditions are overly harsh and causing antigen damage. At the same time, adjust the staining order: schedule targets that are more sensitive to conditions or have weaker expression in earlier rounds.
3) Spectral overlap or bleed-through between channels
When spectral overlap or signal bleed-through occurs between channels, redesign the fluorophore combination. High-abundance targets should be assigned to channels with relatively separated spectra. During imaging and analysis, carefully optimize exposure settings and channel separation strategies to minimize interference.
TSA-based mIHC multiplex fluorescent staining provides a high-sensitivity, high-stability solution for achieving multiplex spatial detection on a single tissue section. Aladdin’s 3–9 color multiplex fluorescent staining kits have been systematically configured in terms of channel number and secondary antibody systems. They are suitable both for laboratories that are newly adopting multiplex staining and wish to gradually establish three-color and four-color panels, and for teams with mature antibody systems that aim to rapidly expand to six-color or even nine-color high-dimensional panels.By optimizing antigen retrieval, primary antibody concentration, and staining order on the basis of single-plex assays, and by selecting the appropriate anti-rabbit or rabbit/mouse universal secondary antibody kit, a robust and reproducible mIHC workflow can be established, providing reliable spatial information support for tumor immune microenvironment profiling, disease subtyping, and studies of drug mechanisms of action.
Aladdin: https://www.aladdinsci.com/