Tyramide Signal Amplification (TSA) Applications

Tyramide signal amplification, or TSA, is also known as catalyzed reported deposition (CARD) and tyramine amplification technique (TAT). It is used for the detection of biological molecules such as proteins, DNA, and viruses within a sample.

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TSA has been applied to improve immunohistochemistry, in situ hybridization and other protocols while using pre-existing imaging techniques, such as microscopes.

Basic methodology

TSA

In TSA, horseradish peroxidase reacts with hydrogen peroxide and the phenolic functional group of tyramine. The result is a tyramine in an activated state with a radical-containing quinone-like structure located on the C2 group, which covalently binds to tyrosine residues of nearby protein molecules.

The amplification is carried out by the activated, radical tyramine molecule, whereupon binding confers a fluorescent signal. The method has good amplification effect and is simple to conduct, making it widely applied with other methods.

TSA-FISH

TSA is often used with fluorescence in situ hybridization (FISH), more commonly referred to as CARD-FISH. In FISH, labeled probes are added to a sample and hybridize to their biological targets inside the cells. This prepares the sample for identification of single cells and quantification by epifluorescence microscopy or flow cytometry.

The growth in the use of TSA-FISH has been facilitated by a modification of the method. The cells are embedded in agarose gel, as this allows for a harsh enzymatic or chemical permeabilization technique to be utilized without the cost of widespread loss of cells by lysis or detachment.

Permeabilization is an important step for TSA-FISH because of the horseradish peroxidase, which is much larger than fluorescent dye molecules, necessitating permeabilization.

The TSA-FISH technique allows for the identification of more cells compared with TSA. A general bacterial probe in the surface water of the North Sea could identify 80% of cells, compared to the below 50% previously achieved by TSA.

TSA-FISH has superior sensitivity to other methods, making it suitable for the simultaneous detection of mRNA and rRNA from bacteria in the environment. This facilitates linking the identification of single cells to expression of certain genes.

Applications in cancer

Sialic acid is found on the terminal sugar chains of glycolipids and glycoproteins. It has recently gained traction as a different tumor-related antigen. It is different due to it displaying strong antigenicity, or its capacity to bind with products that have adaptive immunity, such as T cell receptors or antibodies.

TSA has been successfully applied in the detection of this antigen in tumor tissues from patients with liver cancer. Further, TSA methodology found that the presence of certain antibodies, IgC and IgM, was attributed to sialic acid expression. Therefore, TSA has been found to be useful for immunohistochemical detection of sialic acid expression in carcinoma cells.

Intracellular kinase cascades

Intracellular flow cytometry has often been employed to measure the quantity of a wide variety of molecular targets at the single cell level. However, the detection sensitivity has proven to be inherently limited, which can hinder the measurement of low abundance proteins or the identification of cells expressing slightly different protein concentrations.

TSA allows for the detection of low abundance intracellular proteins. Amplification by TSA improves the resolution of the assay by 30-fold, and allows for improved sensitivity of measurement in comparison to conventional staining methods.

Furthermore, the use of TSA exposed functional heterogeneity within immune cell populations by cytokine stimulation and downstream Stat1 measurements.

This could potentially lead to the identification of new cell types as characterization can be refined. The types of tyramides were also evaluated in this study, and it was found that the best tyramides were those with low non-specific intracellular binding.

Further Reading

• All Flow Cytometry Content
• Flow Cytometry Methodology, Uses, and Data Analysis
• Flow Cytometry Techniques used in Medicine and Research
• Flow Cytometry History
• Fluorescence-Activated Cell Sorting

Written by

Sara Ryding

Sara is a passionate life sciences writer who specializes in zoology and ornithology. She is currently completing a Ph.D. at Deakin University in Australia which focuses on how the beaks of birds change with global warming.