This tyramide signal amplification (TSA) protocol can be used to detect signal in peroxidase-labeled specimens in both immunostainings and in situ hybridization. For immunostaining, it is easier to get TSA to work since the signal is much stronger than in situ hybridization. For in situ hybridization, many rounds of trial and error are expected before one can get a reasonably good staining.

1. Following peroxidase-conjugated antibody labeling and antibody washes, specimens were washed twice in 100 mM borate pH 8.5 supplemented with 0.1% Tween-20, 3 minutes each.

2. Assemble the TSA reaction buffer immediately before use:

3. 100 mM borate, pH 8.5, 0.1% Tween-20, 0.003 % H2O2 (1:10,000 dilution from 30% stock by 2X 1:100 serial dilution)

4. Optional additives for enhancing sensitivity (can work alone or in combination): (1) 2% dextran sulfate (DS) [crowding reagent; dissolve 1g DS in 50 mL 100 mM borate, pH 8.5, 0.1% Tween-20. Take an appropriate amount of DS-borate buffer (e.g. 100 μL per tube of specimen) and add 0.003% H₂O₂ (and 500 μg/mL 4-iodophenol, optional) immediately before use]; (2) 500 μg/mL 4-iodophenol [peroxidase reaction accelerator; 1:200 dilution of 100 mg/mL stock solution (in EtOH)]

5. Add 0.5-2 μL of TSA stock (start with 1 μL, optimal concentration needs to be determined empirically for each case) to 100 μL of TSA reaction buffer. Mix by vortexing.

6. Incubate the specimen with TSA working solution in dark at room temperature for 15-30 min.

7. 3X quick wash with PBS + 0.1% Tween-20 (PBT), followed by several 30 mins wash in PBT and an overnight PBT wash on rocking table at room temperature. All washes are carried out in dark.

8. Check the fluorescence background under a fluorescence microscope. Continue to wash if the background is high (by comparing to the pattern obtained with single-probe AP coloration whole-mount in situ hybridization). Stop wash if the background is low enough or could not be further reduced by overnight wash.

9. If there will be another antibody labeling-TSA reaction cycle, treat the specimen with acid glycine buffer (0.1 M glycine pH 2, 0.1% Tween-20) for 10 min at room temperature.

10. The TSA-labeled specimen can be viewed in any mounting medium since the labeling is covalently linked to proteins in the specimen.

TSA synthesis

Commercial TSA kits are expensive and the fluorescent tyramide is usually too diluted for visualizing gene expression by whole-mount in situ hybridization in early Helobdella embryos. We synthesize our own homemade high-concentration TSA solution for fluorescent in situ hybridization.

1. Prepare the following stock solutions immediately before synthesis. DO NOT STORE these solutions since humidity caught up during storage would hamper cross-linking reaction between fluorophore/hapten and tyramine. Also make sure that N,N-dimethylformamide (DMF) is fresh from the sealed container to avoid humidity contamination. Anhydrous DMF packed in Sure/Seal™ bottle (Sigma 227056) is recommended for this purpose. For best results, carry out synthesis reaction in low humidity environment.

2. 10 mg tyramine (Sigma T2879; MW 173.64) in 990 μL water-free DMF + 10 μL triethylamine (Sigma T0886)

3. 10 mg amine-relative fluorophore or hapten in 1 mL of water-free DMF. Amine-reactive compounds are available from Molecular Probes (Life Technologies) and Pierce (Thermo).

4. Some fluorophores/haptens that can be used for synthesizing labeled tyramide are given in Table 1 below; this list is non-exclusive.

5. Mix tyramine solution and amine-reactive fluorophore/hapten at a 1:1.1 molar ratio. Incubate the mixture in dark for 2 hours. See Table 1 for specific amounts used for each fluorophore/hapten.

6. Add an appropriate amount of ethanol to the mixture so the final concentration of fluorophore/hapten in the stock solution is 5 mg/mL. Mix well, and aliquot. Store the aliquots in -20°C.

Table 1 Reaction setups for TSA synthesis