Tissue microarray five-color immunofluorescence (IF) staining (TSA method)

Service introduction:

Multiplex immunohistochemistry (mIHC) is an important technique that has emerged in the field of pathology research in recent years. This method is a multiplex sequential immunostaining technique based on tyramide signal amplification (TSA), which can detect multiple targets in situ in cell or tissue samples. By studying the combinations and spatial relationships of these targets, their interaction mechanisms can be elucidated. This technique not only substantially improves the sensitivity of detected signals, but also solves the challenge of detecting multiple biomarkers from the same host species on the same section. Through quantitative pathology analysis, information such as the types, components, and expression levels of various targets in situ in tissues and cells, as well as the spatial localization, 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.

Using the TSA method, this project can overcome the limitation of primary antibody host species and label five antigens on a single tissue microarray, without the need to consider whether the primary antibodies are derived 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. Flexible combination of different targets, multi-marker colocalization staining, and the ability to separate signals.

4. Display spatial relationships and distinguish different cell subsets.

Detection principle:

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

Experimental workflow:

Rigorous experimental design for TSA-based multicolor immunofluorescence:

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

2. IF immunofluorescence single-marker pilot experiment (TSA method) - validation of concentrations, adjustment and optimization of color combinations - customer confirmation of results (expression abundance).