Signal Amplification Using the TSA Biotin System

1. For each test, dilute the stock solution of biotinyl tyramide 1:50 with 1X amplification diluent to prepare the working solution; 100 to 300 pL of working solution are needed for each slide.

2. After hybridization, block slides by incubation with 100 to 300 pL of TNB buffer. This may be performed for 30 min in the humidified chamber of the cyler with biotin-labeled probes: 100 to 300 pL of SA-HRP (streptavidin-horseradish peroxidase from the TSA kit) diluted 1:100 in TNB buffer. Fluorescein-labeled probes may be substituted: 100 to 300 pL of anti-fluorescein-HRP (NEN) diluted 1:250 in TNB buffer. Optimal concentrations of HRP-labeled reagents should be determined for individual laboratories.

3. Wash slides in TNT buffer with agitation three times for 5 min at room temperature.

4. Pipet 100 to 300 pL of the working solution onto each slide. Maintain slides at room temperature for 5 to 10 min.

5. Repeat washing step 3.

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