Tyramide Signal Amplification in Planarians

Tyramide conjugates were synthesized as described [49 (link)] from N-hydroxy-succinimidyl-esters of 5/6-carboxyfluorescein (Pierce), 5-(and-6)-carboxytetramethylrhodamine (Molecular Probes), DyLight 633 (Pierce), and 6-(2,4-dinitrophenyl) amino hexanoic acid (Molecular probes). Tyramide signal amplification was performed by incubating planarians for 10 min in fluorophore-conjugated tyramide diluted 1:250–1:500 in 100 mM borate buffer pH 8.5, 2 M NaCl, 0.003% H₂O₂, and 20 μg/ml 4-iodophenylboronic acid. For double FISH experiments, residual peroxidase activity was quenched by incubating for 45 minutes in 100 mM glycine pH 2.0 or in PBSTx containing either 2% H₂O₂, 4% formaldehyde, or 100 mM sodium azide.