TSA-mIHC multiplex fluorescence immunohistochemistry technology has become one of the core technologies in current pathological research due to its advantages of ultra-high sensitivity, simultaneous multi-color chromogenesis, and high specificity.

As the "signal converter" of ELISA detection, the quality of TMB chromogenic components directly determines the accuracy of OD values, and even more determines the reliability and repeatability of final experimental data. Understanding the core value of TMB is the only way to truly break out of the experimental error misunderstanding and achieve the transformation from "measuring values" to "measuring accurate data".

Zhang Yao et al.'s article "Single-Cell Analysis of Chemotherapy-induced Remodeling Reveals CD276-driven Basal-like Chemoresistance in Pancreatic Cancer," published in the 2026 issue of Gastroenterology, leverages high-precision multi-omics technology to focus on chemotherapy-induced dynamic remodeling of PDAC tumor cells and microenvironment, deeply excavating the core molecular mechanisms of chemotherapy resistance, aiming to solve the dilemma of PDAC chemotherapy resistance and provide new targets and theoretical support for clinical precision targeted therapy.

In life science research, antibody labeling is a very common and important technique. By conjugating antibodies with fluorescent dyes, enzymes, luminescent groups, or other functional molecules, antibodies can be endowed with new detection capabilities, making them widely used in experiments such as immunofluorescence (IF), flow cytometry (FCM), immunohistochemistry (IHC), Western blot, ELISA, and protein localization analysis.

In the world of biomedical research, clinical diagnostics, and drug development, antibodies are the "exclusive detectors" that precisely identify specific antigens. However, antibodies themselves cannot be directly captured by the naked eye or instruments. To make their recognition process "visualizable, quantifiable, and traceable," antibodies must be bound with special "signal tags" — this is the core principle of antibody labeling technology.

Why is TMB initially colorless? Why does it definitely turn blue after catalysis? Why does stopping the reaction specifically turn it yellow? Today, we will set aside obscure formulas and break down the complete principles of TMB color development reaction in an easy-to-understand way, revealing the chemical essence behind the experimental phenomena.

Tyramide Signal Amplification (TSA) is an enzyme‑catalyzed in situ detection method that overcomes the sensitivity and multiplexing limitations of conventional immunofluorescence. The core mechanism relies on the high catalytic activity of horseradish peroxidase (HRP) to achieve specific, high‑density labeling of target proteins and nucleic acids.

Currently, phycobiliproteins have become irreplaceable fluorescent labeling systems in flow cytometry, multiplex immunofluorescence, immunohistochemistry, and immunoassay platforms, with phycoerythrin (PE) and allophycocyanin (APC) being the most widely used.

Fluorescent labeling technology is one of the most important visualization tools in modern life science research. From intracellular protein localization and gene expression monitoring to immune cell typing, multi-parameter flow analysis, and spatial biology research, fluorescent signals are almost ubiquitous throughout the life science experimental workflow.

We have conducted extensive multiplex TSA staining on paraffin samples in our daily work, encountered many pitfalls, and developed a stable experimental protocol. Below, we summarize and share our practical experience, hoping to help everyone avoid detours and improve experimental success rates.