From Literature: Understanding How TSA Technology Empowers mIHC Multiplex Immunofluorescence to Overcome Detection Limitations

The study used the TSA tyramide amplification system, direct fluorescent secondary antibody conjugation system, and streptavidin direct-labeled fluorescence system to detect CD31, a mouse embryonic vascular endothelial marker. In large blood vessels with high CD31 expression, all three detection methods could detect clear signals, and the differences were not obvious. However, when facing microvessels and low-expression target protein regions that are common in organisms, the differences were completely revealed. The two traditional fluorescence detection methods could not capture weak specific signals at all; the microvessel structures were completely submerged by background fluorescence, and no effective positive signals could be identified. In contrast, the experimental group equipped with TSA signal amplification, relying on the enzymatic reaction to accumulate a large number of fluorescent tyramide molecules at antigen binding sites, efficiently amplified the originally invisible weak signals, completely outlining the fine microvascular network. At the same time, the autofluorescence levels of red blood cells in each group were completely consistent, indicating that TSA only specifically amplifies specific target signals without increasing non-specific background noise, perfectly balancing the two advantages of high sensitivity and low background, which is a technical breakthrough that all traditional fluorescence detection systems cannot achieve.

Spleen immune cell subtypes are complex, and multiplex staining is prone to signal overlap and non-specific antibody binding. The study used TSA technology to perform CD3, CD4, and CD8 triple-label fluorescence staining on human spleen sections, simultaneously distinguishing different T cell subsets. The results showed that relying on the TSA round-by-round signal amplification mode, the three fluorescence signals had clear boundaries without mutual interference. CD4 and CD8 positive cells had completely separate spatial distributions, allowing precise distinction between the two T cell subsets. At the same time, both cell types perfectly co-localized with the universal T cell marker CD3. Different from traditional multiplex fluorescence staining, where signals gradually attenuate and crosstalk becomes more severe as the number of markers increases, each round of TSA staining can independently complete signal amplification and fixation, ensuring that each target signal has sufficient fluorescence intensity. Even after three rounds of sequential staining, extremely high signal specificity can still be maintained, proving that TSA can stably support multi-target in-situ co-localization analysis in complex tissues.

Plasma cells in mouse spleen naturally contain a large amount of endogenous immunoglobulins, which directly bind to secondary antibodies, producing inherent non-specific background. This type of background is an inherent interference of the tissue itself and cannot be eliminated by optimizing the staining process. The experimental results showed that even with this inherent background interference, TSA still did not amplify non-specific endogenous background signals, only specifically amplifying fluorescence signals at antigen-antibody specific binding sites. The specific vascular signals in the experimental group were clear and bright, while the non-specific plasma cell fluorescence signals always maintained their original weak levels and would not be synchronously amplified to aggravate background interference. This result proves that TSA has the core advantage of targeted site-specific amplification, does not non-selectively amplify all fluorescence signals on the entire section, and maximally retains the difference between effective signals and background signals, ensuring that the signal-to-noise ratio of staining results is always at a high level.

Due to antibody compatibility and signal interference issues, traditional immunofluorescence can achieve at most triple-label detection, and cannot use same-species primary antibodies, which greatly limits multi-dimensional molecular analysis of the tumor microenvironment. To break through this detection throughput bottleneck, this study relied on TSA technology to simultaneously detect four markers in human kidney tumor sections, including three mouse homologous primary antibodies. By completely inactivating the HRP enzyme activity of the previous round after each round of staining, TSA blocks cross-reactions between antibodies from different rounds. Finally, the four fluorescence signals did not interfere with each other, clearly distinguishing four cell populations in the tumor tissue: vascular endothelial cells, stromal cells, epithelial cells, and proliferating cells, and clearly delineating the junction area between tumor and normal tissue. It is sufficient to illustrate that TSA has completely broken two major constraints of traditional fluorescence staining: first, it breaks through antibody species restrictions, allowing free combination of homologous primary antibodies; second, it breaks through the limit of marker number, supporting quadruple-label and above ultra-high-throughput multiplex detection, significantly increasing the detection information content of a single tissue section.

In previous multiplex fluorescence experiments, primary antibodies from different species must be used. Once same-species antibodies are used, subsequent secondary antibodies will non-selectively bind to all primary antibodies, causing comprehensive non-specific staining. This study used TSA combined with antigen saturation blocking treatment, using 3 rabbit primary antibodies to detect mouse ovary and 2 mouse primary antibodies to detect mouse liver, and finally no cross-binding phenomenon was observed in the staining results. The signals from single-channel independent staining completely matched those from multi-channel overlay, and the two homologous antibodies did not interfere with each other, with staining patterns identical to single-label staining. Relying on the enzymatic signal immobilization effect of TSA, antigen sites are completely blocked after each round of staining, preventing binding with homologous antibodies in the next round, completely liberating antibody selection restrictions. This is an advantage that cannot be achieved by all traditional multiplex staining methods without TSA support.

Mouse embryonic tissues have fast cell differentiation, fine tissue structure, and extremely high complexity. Conventional fluorescence staining is prone to problems such as signal loss and uneven staining. In contrast, the automated staining system based on TSA, whether for embryonic quadruple-label organ distribution detection, triple-label cell proliferation detection, or double-label metabolic activity detection, can output high signal-to-noise ratio and high-definition fluorescence images, enabling accurate identification of target molecules in the fine tissue structures of embryos. At the same time, this technology is compatible with various samples such as paraffin sections, frozen sections, and cultured cells, adapting to both clinical pathological samples and basic research samples. Compared with the strict sample requirements of traditional staining methods, TSA technology has a wider range of experimental applications and adapts to the needs of in-situ molecular detection of all types of tissues.

Compared with the immunohistochemistry system, TSA technology applied to immunofluorescence can also maintain a linear relationship between fluorescence signal intensity and target protein expression, with the ability for precise quantitative analysis. Overall, TSA technology has shown significant advantages in sensitivity, antibody compatibility, detection throughput, experimental repeatability, and quantitative ability, and has become an indispensable core technology in tissue multiplex immunofluorescence detection.

Yarilin D, Xu K, Turkekul M, Fan N, Romin Y, Fijisawa S, Barlas A, Manova-Todorova K. Machine-based method for multiplex in situ molecular characterization of tissues by immunofluorescence detection. Sci Rep. 2015 Mar 31; 5:9534. doi: 10.1038/srep09534. PMID: 25826597; PMCID: PMC4821037.

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