Overview
This lecture covers the Tyramide Amplification Kit with HRP Goat Anti-Mouse and CF Dye or Biotin Tyramide, focusing on its use in increasing immunofluorescence sensitivity via tyramide signal amplification (TSA).
Tyramide Signal Amplification (TSA) Principle
- TSA uses HRP enzyme activity to deposit dense labels at the target protein/nucleic acid site.
- Also known as Catalyzed Reporter Deposition (CARD).
- Can increase sensitivity up to 100-fold over standard avidin-biotin methods.
Kit Features
- Allows use of lower concentrations of primary antibody.
- Greatly enhances signal in challenging samples (e.g., FFPE tissue).
- Offers 6 bright, photostable CF dyes or biotin as labeling options.
- Each kit includes enough reagents for 50–150 slides (actual amount depends on use).
Kit Components
- Contains CF dye or biotin-tyramide stock solution.
- Includes highly cross-adsorbed HRP goat anti-mouse IgG (minimal cross-reactivity to bovine, horse, human, rabbit, and swine).
- BSA for blocking, Tyramide Amplification Buffer Plus, and 30% hydrogen peroxide included.
Applications and Variants
- Multiplex detection possible by multiple rounds of amplification.
- Kits are available with other secondary antibodies: goat anti-rabbit HRP and HRP-streptavidin.
- CF dyes offer superior brightness and photostability for fluorescence imaging.
Storage and Handling
- Store kit at -10°C to -35°C.
- Protect dye component from light to maintain performance.
Dye/Conjugate Options
- Dye options: CF488A (490/515 nm), CF543 (541/560 nm), CF568 (562/583 nm), CF594 (593/614 nm), CF640R (642/662 nm), CF680R (680/701 nm), Biotin-XX.
- Conjugates offered: Goat anti-mouse HRP, goat anti-rabbit HRP, and streptavidin HRP.
Key Terms & Definitions
- Tyramide Signal Amplification (TSA) — An HRP-catalyzed labeling method for boosting immunofluorescence signals.
- HRP (Horseradish Peroxidase) — An enzyme that catalyzes tyramide deposition, enabling signal amplification.
- CF Dyes — Next-generation, highly photostable and bright fluorescent dyes.
- FFPE — Formalin-fixed, paraffin-embedded tissues, often challenging for immunofluorescence.
Action Items / Next Steps
- Review [Tech Tip: Multicolor Fluorescence Imaging using Tyramide Amplification Kits].
- Learn about [CF dyes] and [tyramide signal amplification] for in-depth understanding.
- Ensure proper storage (-10°C to -35°C, protect from light) of kit components.