This protocol provides the sensitivity of enzymatic detection (HRP) in immunohistochemical procedures with the versatility and convenience of fluorescence detection. Tyramide amplification was first developed at DuPont NEN by Litt’s group (Bobrow et al. 1989, J. Imm. Meth., 125: 279-285; Bobrow et al. 1991, J. Imm. Meth. 137: 103-112) and has since been made into an off-the-shelf kit called "TSA" for tyramide signal amplification. I have included here the use of the TSA kit as well the the synthesis of the tyramide reagents and the recipe for the reaction buffer. Synthesis of these reagents in house is not only simple but can potentially save a lot of money. For approximately $300.00 a single TSA kit can be purchased for one fluorochrome that is enough for approximately 75-100 slides. For that same amount of money, reagents can be purchased to stain over 1,000,000 slides using two different fluorochromes.

0.1 M Borate 8.5 6.18 g boric acid

up to 1 liter with Q, pH to 8.5 with NaOH

per liter (Sigma #H1009)

Amplification Diluent can be stored at 4°C for several months. I suggest that you make the borate buffer stock and then add hydrogen peroxide to a smaller volume (100 ml) for daily use.

Tyramine HCl (5g), Aldrich #T9,035-2

5-(and-6)carboxyfluorescein, succinimidyl ester (100 mg), Molecular Probes #C-1311

Texas Red succinimidyl ester is available from Molecular Probes

Cy-3 succinimidyl ester is available from Amersham

Biotin, succinimidyl ester is available Calbiochem

synthesis of tyramide compounds

• Prepare succinimidyl ester (46.6 mg (FITC)/0.3 ml) and tyramine solutions (14.6 mg/0.3 ml) in DMSO and mix them together at a 1:1 ratio in a light proof bottle at room temperature overnight.

• This tyramine stock can be used at a 1:50,000 dilution. NOTE: for other tyramine compounds calculate the molarity to match that of the FITC, siccinimidyl ester shown above.

• The tyramide compounds are stable for years when stored at 4°C.

amplification

• Dilute the tyramide compound in amplification diluent to 1:50,000 (or 1:150 for DuPont NEN) and add directly to slides after antibody incubations etc...

• Incubate for 10 minutes at room temperature and stop the reaction by washing in PBS.

• DAPI stain and mount with gelvatol.