Fifty mg of sulfocuccinimidyl-6-(biotinimide) hexanoate (NHS-LC-biotin) Pierce; was dissolved in 20 ml of 50mM borate buffer, pH 8.0. Fifteen mg tyramine -HCL Sigma was added. The solution was gently rocked overnight in the dark at RT and filtered through a .2µm filter. One ml aliquots of this biotinylated tyramine (BT) were frozen at -75 Celcius.
Following the application of anti-digoxigenin HRP labeled antibody at a concentration of 1:100 the sections were washed in .1MPBS for 3 x 10 minutes at RT. The sections were incubated in a solution of BT (1:100 to 1:1000) in PBS and .01% H2O2 containing 10% Dextran Sulphate.
Sections were then washed in PBST for 3 x 10 minutes at RT and incubated in ABC for 2 hours at RT. Sections are washed and a standard DAB reaction was performed.
Alternatively, Avidin-coupled flourochromes can be applied at 1:100 for a flourescent signal.