Tyramine is a primary amino compound obtained by formal decarboxylation of the amino acid tyrosine:
Below is the protocol for labeling of tyramine molecule with sulfo-Cyanine3 NHS ester product for use in tyramide signal amplification.
All steps are protected from light and performed at room temperature.
Solution 3: 10 mg/mL Tyramine HCl in Solution 2 (10 mg Tyramine HCL in 1 mL Solution2).
Reaction: Mix together 100 uL of Solution 1 + 27 uL of Solution 3; incubate for 4 h at room temp, then add 1.2 mL 100% EtOH.
Results tested side by side with known working fluorescent tyramide conjugates via FISH with digoxigenin-labeled RNA probes and HRP-conjugated anti-digoxigenin antibodies.
The sulfo-Cyanine3-labeled tyramide was tested at several concentrations and showed positive staining similar to control slides.
At the same time, non-sulfonated Cyanine3-labeled tyramide (Cyanine3 NHS ester was used for labeling) was tested at several concentrations and showed no staining whatsoever, while control slides showed normal staining.