​ Multiplex Immunofluorescence mIHC Kit

Multiplex Immunofluorescence mIHC Kit

I. What is Multiplex Immunofluorescence (mIHC) Kit?

Due to the complex experimental workflow of mIHC, which involves multiple critical steps such as antigen retrieval, signal amplification, fluorescence channel matching, antibody stripping, and background control, professional mIHC kits can effectively improve experimental repeatability, reduce fluorescence crosstalk and non-specific background by integrating optimized fluorescent dye systems, HRP detection reagents, and supporting buffer systems, providing standardized support for high-quality, multi-marker tissue imaging research.

TSA (Tyramide Signal Amplification) kits are high-sensitivity immunoassay systems developed based on the principle of HRP enzyme-catalyzed fluorescent tyramide molecule deposition. Its core mechanism is to use HRP-labeled secondary antibody to catalyze the activation of fluorescent tyramide, causing it to undergo covalent deposition around the target antigen, thereby achieving local high-density fluorescence enrichment and significantly improving detection sensitivity. It is particularly suitable for the detection of low-expression antigens, FFPE tissue samples, and weak signal proteins. Compared with traditional immunofluorescence technology, TSA kits can not only achieve tens of times or even higher signal amplification, but also retain stable fluorescence signals during multiple rounds of staining. Therefore, it supports 2-6 plex or even higher throughput multi-marker detection on the same tissue section, and effectively reduces the limitations caused by cross-reactivity of same-species antibodies. Professional TSA kits usually include key components such as fluorescent tyramide dyes of different wavelengths, HRP secondary antibodies, blocking solutions, antigen retrieval solutions, antibody stripping solutions, and anti-fluorescence quenching mounting media. These components can help researchers reduce background noise, minimize fluorescence crosstalk, improve experimental repeatability and tissue signal stability. Meanwhile, they are compatible with automated staining platforms, spectral imaging systems, and digital pathology analysis software. Therefore, TSA kits have become core tools in tumor immune microenvironment research, PD-1/PD-L1 immunotherapy evaluation, spatial omics analysis, and precision pathology research.

II. Product Advantages of Multiplex Immunofluorescence mIHC Kit

1. High Sensitivity Detection

Based on TSA enzymatic signal amplification technology, the mIHC kit can significantly enhance the fluorescent signal of target proteins. Even for low-abundance, weakly expressed, or difficult-to-detect antigens, clear and stable imaging results can be obtained. It is particularly suitable for FFPE tissue samples and clinical pathological research.

2. High-Throughput Multiplex Detection Capability

Compared with traditional IHC or conventional immunofluorescence which can only detect 1-2 markers at a time, the mIHC kit can achieve 2-7 color or even higher throughput Marker combined detection on the same tissue section. It can simultaneously analyze multiple immune cells, tumor markers, and functional proteins, greatly improving the efficiency of tissue information acquisition.

3. Compatible with FFPE Clinical Samples

Professional mIHC kits are optimized for formalin-fixed paraffin-embedded (FFPE) tissues, which can effectively improve antigen retrieval efficiency and weak signal detection capability. Therefore, they are widely applicable to various tissue samples such as paraffin sections, frozen sections, cell slides, cell smears, and organoids.

4. Complete Preservation of Spatial Information

mIHC technology can not only detect protein expression levels but also preserve the spatial distribution relationship of cells in tissue situ, enabling spatial localization analysis between tumor cells and immune cells, providing important support for spatial pathology and spatial biology research.

5. Stable Signal and Low Background

The optimized TSA system and buffer system can effectively reduce non-specific background, fluorescence crosstalk, and antibody cross-reactivity issues, improve experimental repeatability and data consistency. It also has excellent anti-quenching performance, suitable for long-term scanning and digital pathology analysis.

6. Support for Automation and Digital Pathology Analysis

Modern mIHC kits are usually compatible with automated staining platforms, multispectral scanning systems, and AI digital pathology analysis software, enabling high-throughput, standardized experimental workflows, and supporting single-cell typing, spatial neighborhood analysis, and quantitative pathology research.

III. EnkiLife Multiplex Immunofluorescence mIHC Kit Products

TSA Complete Kit Components

IV. Kit Operation Procedure (Taking Paraffin Sections as Example)

1. Deparaffinization: Soak the sections in Xylene 1 (15min), Xylene 2 (15min), Anhydrous Ethanol 1 (5min), Anhydrous Ethanol 2 (5min), 95% Ethanol (5min), 85% Ethanol (5min), 75% Ethanol (5min) in sequence, and finally rinse the slides with water.

2. Antigen Retrieval: Usually use 1× Citric Acid (PH6.0) as the retrieval solution. Perform high-temperature and high-pressure retrieval by placing the sections in a pressure cooker, adding an appropriate amount of retrieval solution, closing the pressure cooker, timing for 2min after steam generation, cooling to room temperature, and completing the retrieval. 【For markers with weak expression, EDTA (PH9.0) can be used as the retrieval solution for antigen retrieval. For tissues prone to detachment such as bone tissue and brain tissue, microwave retrieval is recommended. The retrieval temperature should be controlled at around 80°C, and 2× Citric Acid (PH6.0) can be used as the retrieval solution.】

3. Endogenous Enzyme Blocking: Prepare 3% H2O2 solution with pure water, place the sections in the solution, and incubate at room temperature for 20min. For tissues prone to detachment, the H2O2 concentration and incubation time can be appropriately reduced.

4. Blocking: Add blocking solution dropwise onto the tissue, and incubate at 37°C for 30min.

5. Primary Antibody Incubation: Dilute the primary antibody to an appropriate concentration using antibody diluent, add the antibody dropwise onto the tissue, and incubate overnight at 4°C, or incubate at 37°C for 1h.

6. Secondary Antibody Incubation: Add HRP enzyme-labeled secondary antibody dropwise onto the tissue, and incubate at 37°C for 1h.

7. TSA Reagent Incubation: Add TSA-XXX fluorescent dye dropwise onto the tissue, incubate at 37°C for 30min, and wash with PBST 3 times, 5min each time.

8. Antibody Stripping: Add antibody stripping solution dropwise onto the tissue, and incubate at room temperature for 15min (incubation at 37°C is better).

9. Repeat steps 4-8 (incubate other TSA-XXX fluorescent dyes)

10. DAPI Nuclear Staining: Add DAPI working solution dropwise onto the tissue, incubate at room temperature for 5min, wash with PBS 3 times, 5min each time. Dry the liquid, add anti-fluorescence quenching mounting medium dropwise onto the tissue, cover with a coverslip, and observe and photograph under a microscope.

EnkiLife focuses on the field of multiplex immunofluorescence and spatial biology research. We can provide research users with complete TSA-mIHC reagent systems, optimized experimental protocols, and professional technical support services, helping researchers quickly establish a stable and reliable multiplex fluorescence detection platform and promoting the realization of high-quality scientific research results and clinical translational research.