Tissue microarray triple immunofluorescence staining (TSA method)

Service introduction:

Multiplex immunohistochemistry/immunofluorescence (mIHC) is an important technology that has emerged in pathology research in recent years. This method is a multiplex sequential immunostaining technique based on tyramide signal amplification (TSA). It can detect multiple targets in situ in cell or tissue samples, and by studying the combination and spatial relationships of these targets, elucidate their interaction mechanisms. This technique not only greatly improves the sensitivity of detected signals, but also solves the challenge of detecting multiple same-species biomarkers on the same section. Through quantitative pathology analysis, information such as the categories, components, and expression levels of various targets in situ in tissues and cells can be obtained, as well as the spatial localization, qualitative and quantitative information of each target and its interactions. Comprehensive, systematic, intuitive, and scientific interpretation of this information is of great significance for studying the mechanisms of disease occurrence and progression, and for exploring effective methods for disease diagnosis and treatment.

In this project, the TSA method can overcome the limitation of primary antibody species, allowing three antigens to be labeled on a single tissue microarray without the need to consider whether the primary antibodies are from the same species.

Main applications:

1. Dual advantages of in situ visualization and quantitative analysis.

2. More comprehensive analysis: simultaneous analysis of drug targets and the tumor microenvironment.

3. Free combination of different targets, multi-target co-localization staining, and signal separation.

4. Demonstration of spatial positional relationships and distinction of different cell subsets.

Detection principle:

In the presence of hydrogen peroxide, enzymes such as horseradish peroxidase and alkaline phosphatase can catalyze a tyramide substrate into a highly reactive, short-lived intermediate. Within a short time, the intermediate can covalently bind to electron-rich surfaces of surrounding proteins to form tyramide complexes. Because the adjacent proteins (such as HRP, antibodies, and target antigens) contain abundant tyrosine binding sites, a large amount of signal is enriched, thereby effectively amplifying the signal.

Experimental workflow:

Strict experimental design for TSA-based multiplex immunofluorescence:

1. IHC immunohistochemistry pilot experiment - antibody validation - customer confirmation of results.

2. IF immunofluorescence single-label pilot experiment (TSA method) - validation of concentration and color combination adjustment and optimization - customer confirmation of results (expression abundance).