Tissue microarray immunofluorescence (IF) quadruple labeling (TSA method)

Service introduction:

Multiplex immunohistochemical labeling technology (mIHC) is an important technical method that has emerged in the field of pathology research in recent years. This method is a multiplex sequential immunohistochemical staining technique based on tyramide signal amplification (Tyramide Signal Amplification, TSA). It can detect multiple targets in situ in cell or tissue samples, and through studying the combinations and positional relationships of these targets, their interaction mechanisms can be elucidated. This technology can not only greatly improve the sensitivity of detected signals, but also solve the challenge of detecting multiple biomarkers from the same species on the same section. Through quantitative pathological analysis, information such as the types, components, and expression levels of various targets in situ in tissue cells, as well as the spatial localization and qualitative and quantitative information of each target and its interactions, can be obtained. 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 imposed by the species origin of primary antibodies, label four antigens on a single tissue microarray, and eliminate 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, multiplex co-localization staining, and the ability to separate signals.

4. Reveal spatial positional relationships and distinguish different cell subpopulations.

Detection principle:

In the presence of hydrogen peroxide, certain enzymes such as horseradish peroxidase (HRP) 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 the electron-rich surfaces of surrounding proteins to form tyramide complexes. Because the associated proteins (such as HRP, antibodies, and target antigens) contain abundant tyrosine residues, large amounts of signal are deposited, thereby effectively amplifying the signal.

Experimental procedure:

Strict experimental design for TSA-based multiplex immunofluorescence:

1. IHC immunohistochemical preliminary experiment - validate antibodies - client confirms the results.

2. IF immunofluorescence single-label preliminary experiment (TSA method) - verify concentrations and optimize color combination adjustments - client confirms the results (expression abundance).