In the field of biomedical research, multiplex immunohistochemistry (mIHC) technology is becoming increasingly important. This technique allows for the simultaneous detection of multiple proteins on a single tissue section, providing a powerful tool to reveal the complex biological information within cells and tissues. As mIHC technology continues to evolve, more researchers are seeking to visualize a richer array of targets on their tissue sections, which means employing a greater variety of fluorescent dyes for staining. However, the signals generated by fluorescent dyes are not purely linear but rather exhibit a bell-shaped distribution. While the signal is optimal at the peak, it can still be captured within a range around the peak. Consequently, adjacent dyes may overlap in their signals, leading to inaccurate interpretation of the results. So, how can we address this issue?

 

 

Firstly, selecting appropriate dyes is crucial. It is essential to distinguish between excitation and emission wavelengths without enhancing background fluorescence. Choosing dyes with widely separated wavelengths is key to avoiding signal overlap. For example, in the multicolor kits provided by Absin, the dye combinations of four colors (TSA520, TSA570, TSA650, DAPI), five colors (TSA520, TSA570, TSA620, TSA700, DAPI), and seven colors plus (TSA480, TSA520, TSA570, TSA620, TSA700, TSA770, DAPI) exhibit minimal crosstalk.

 

Secondly, optimizing the staining order is vital. We recommend starting with the lowest abundance targets (paired with the brightest fluorescent dyes) and ending with the highest abundance targets (paired with dimmer dyes). Additionally, adjusting primary antibody concentrations, reducing fluorescent dye concentrations, or altering the staining order of antibodies can balance the dye combinations for each target.

 

The principle of dye pairing generally follows “pairing low-expressing targets with strong signals and high-expressing targets with weak signals”. This means that indicators with weaker expression should be paired with dyes that have stronger fluorescent signals, while indicators with stronger expression should be paired with dyes that have weaker signals. Absin's TSA dyes have stronger fluorescence with shorter wavelengths. For example, if we need to stain PD-1, CD8, and CD3 using a four-color kit (TSA520/TSA570/TSA650), IHC results show that PD-1 has very low signal, CD3 has a high signal, and CD8 is intermediate. Therefore, it is reasonable to pair TSA520 with PD-1, TSA650 with CD3, and TSA570 with CD8.

 

Next is how to arrange the staining order:

 

1) Determine cellular localization, from outside to inside: Identify the cellular localization of each marker. Membrane expression should be stained first, followed by cytoplasmic or nuclear staining (if there are many markers, intracellular markers can be stained later without permeabilization after several rounds of washing; if there are few markers, permeabilization is still needed before staining intracellular markers, and multiple intracellular markers only require one permeabilization).

2) Determine antigen retrieval method, acid before alkali: Alkali retrieval is stronger than acid retrieval. Some antibodies may show non-specific staining after alkali retrieval. Therefore, if the cellular localization is the same, stain with acid-retrieved antibodies first, followed by alkali-retrieved antibodies.

3) Based on IHC results, stain weak markers first: Markers that show fewer and weaker signals in IHC should be stained first. Theoretically, markers stained earlier will have results closer to single-label staining.

4) Based on the type of antigen markers: Primary antibody types (difficulty of elution: structural membrane proteins and cytoskeletal proteins > cytoplasmic proteins > nuclear proteins). Unless some antibodies, such as E-cadherin, EpCAM, and Tuj1, bind very strongly to antigens or have elution issues, the primary antibody concentration can be reduced, elution time can be increased, or these antibodies can be placed in the last round of TSA labeling. In experimental design, we also need to consider the specificity and cross-reactivity of antibodies. Using highly validated antibodies ensures reliable results. Generally, antibodies validated for IHC-P can also be used in mIHC, but the experimental steps need to be optimized.

 

Additionally, our multicolor technology at Absin utilizes Tyramide Signal Amplification (TSA) technology, which allows for the co-staining of multiple target proteins on a single tissue section, with the capability of labeling up to tens of proteins simultaneously. A key advantage of TSA technology is the ability to use multiple primary antibodies from the same species without worrying about cross-interactions, greatly simplifying the design process for multiplex detection.

Figure 1: Principle of Tyramide Signal Amplification (TSA) Technology


Figure 2: Workflow of Multiplex Fluorescence Immunohistochemistry (mIHC) Staining

Catalog Number	Product Name	Specifications
abs50015	Absin 7-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50031	Absin 7-Color IHC Kit(Anti-Rabbit Secondary Antibody)	20T/100T
abs50038	Absin 7-Color IHC Kit (plus) (Anti-Rabbit Secondary Antibody)	20T/100T
abs50014	Absin 6-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50049	Absin 6-Color IHC Kit (plus) (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50030	Absin 6-Color IHC Kit (Anti-Rabbit Secondary Antibody)	20T/100T
abs50048	Absin 6-Color mlHC Kit(plus) (Anti-Rabbit Secondary Antibody)	20T/100T
abs50013	Absin 5-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50029	Absin 5-Color IHC Kit (Anti-Rabbit Secondary Antibody)	20T/100T
abs50012	Absin 4-Color IHC Kit (Anti-Rabbit and Mouse Secondary Antibody)	20T/100T
abs50028	Absin 4-Color IHC Kit(Anti-Rabbit Secondary Antibody)	20T/100T

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

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