Post-incubation Heating Significantly Improves Tyramide Signal Amplification

Tyramide signal amplification is based on peroxidation of tyramine and on the high substantivity of the enzymatic product, which binds to nearby protein residues, mainly to tryptophan, histidine, and tyrosine residues (Bobrow et al. 1989, 1992). This principle has been extensively used in immunohistochemistry to enhance target amplification through the in situ deposition of high amounts of biotin-labeled tyramine (tyramide), which are then revealed by peroxidated avidin and diaminobenzidine (DAB)–H₂O₂ incubation or similar detection systems.

The yield in terms of target amplification has allowed a manyfold dilution of primary antibodies (von Wasielewsky et al. 1997) and, in in situ hybridization (ISH), a sensitivity comparable to that reached by use of radioactive probes (Komminoth and Werner 1997; Speel et al. 1997). However, such an increase in sensitivity might be biased by a loss of specificity, and the possibility of an unexpected signal due to unspecific tyramide deposition has been debated (Reubi et al. 1998).

We wish to report here a chance observation that resulted in a marked improvement of the catalyzed reporter deposition (CARD) technique. We observed that heating after incubation with biotinyl–tyramine–H₂O₂ results in a higher intensity of the final staining without loss of specificity.

In standard immunocytochemical procedures, we incubated formalin-fixed, paraffin-embedded tissue sections with a spectrum of routinely employed antibodies. Tissue specimens of human pancreas, large intestine, breast, and placenta were tested with monoclonal mouse antibodies (MAbs) against, respectively, chromogranin A (CgA) (Biogenex; San Ramon, CA), α-smooth muscle actin (αSMA) (Dako; Glostrup, Denmark), S-100 protein (Dako), large-spectrum keratins (KL1; Immunotech, Marseille, France), and human chorionic gonadotropin (βHCG) (Dako). Antigen retrieval procedures (heating in microwave oven for three cycles of 3 min each in citrate buffer) were applied for cytokeratin only.

For the secondary revelation step, the LSAB2 kit (Dako) was employed.

Compared to standard conditions, the use of CARD allowed a manyfold dilution of primary MAbs (von Wasielewsky et al. 1997). At very high dilutions (1:50,000 for CgA and for αSMA, 1:15,000 for KL1, 1:350,000 for S-100, and 1:3000 for βHCG) a weak but still detectable signal was observed on the specific targets.

The standard CARD procedure involves (a) incubation with biotinyl–tyramine–H₂O₂ medium for 15 min, (b) washing twice in PBS for 5 min each, (c) incubation with peroxidated avidin for 15 min, and (d) incubation with DAB–H₂O₂ for 5 min. All these steps are performed at room temperature.

We observed that when the temperature of the washing step (b) was increased to 100C, the final result was markedly stronger (Table 1; Figure 1), and for some antibodies the background resulted decreased. No substantial differences were found among different methods of heating (hotplate, hot buffers, or microwave) or different washing buffers (Tris, pH 8.3, EDTA, pH 8.5, citrate, pH 6, or PBS, pH 7.5). Testing different temperatures, we observed a progressive increase of staining from 60C upwards, with 95–100C showing the strongest result. We have therefore adopted as a standard procedure the sequence outlined in Table 2.

The interpretation of the present findings is uncertain but may be related to a stabilization of the binding of tyramide to electron-rich moieties of proteins, preventing washing-out of the reaction product. An alternative interpretation might be the reduction of unstable dimeric products of biotin–tyramine or a better exposure of biotin. In fact, it has already been proven that heating retrieves biotin reactivity (Bussolati et al. 1997).

Table 1

Heat-induced improvement of tyramide amplification in immunohistochemistry a

Antibody	Source and standard dilution	Dilution rate using standard CARD amplification (von Wasielewsky et al. 1997)	Heat-induced tyramide amplification
Chromogranin A	Biogenex: 1:300	nc	1:50,000
α-Smooth muscle actin	Dako: 1:150	1:12,000	1:50,000 b
Pancytokeratin (KL1)	Immunotech: 1:50	1:500	1:15,000
S-100	Dako: 1:350	1:8750	1:350,000 b
β-HCG	Dako: 1:300	nc	1:3000

^anc, not comparable because not tested by the authors cited.

^bAn additional reduction of background was seen.

Figure 1

Increased sensitivity in immunohistochemistry by means of post-incubation heating in CARD amplification. (a) Chromogranin A (diluted 1:50,000) in pancreatic islet of Langherans. Bar = 75 μm. (b) S-100 (diluted 1:350,000) staining in neural structures of the intestinal wall. Bar = 37 μm. (c) α-Smooth muscle actin (diluted 1:50,000) in small submucosal vessels of the appendix. Bars = 56 μm.

In conclusion, we suggest that, once the best conditions are found for each antibody, the use of postincubation heating in CARD amplification can be considered an easy and useful standard step in immunohistochemistry, both to improve sensitivity and to reduce unspecific background.

Table 2

Schematic sequence of post-incubation heating application in CARD

1. Standard deparaffinization 2. Quenching of endogenous peroxidase activity in H2O2–methanol 3. Antigen retrieval (when applied) Endogenous biotin blocking for 15 min with egg white and 15 min in 0.2% biotin (Sigma; St Louis, MO) in PBS 4. Incubation with primary Ab for 30 min at RT 5. Secondary Ab incubation for 10 min followed by streptavidin incubation for 10 min (LSAB2 kit; Dako) 6. Incubation in biotinylated tyramide–H2O2 for 15 min (Dako GenPoint; Dako) 7. Washing for 5 min in hot (95 C) PBS buffer 8. Washing for 5 min in PBS at RT 9. Streptavidin incubation for 10 min (Dako) 10. Development in DAB H2O2 solution (Sigma) for 5 min 11. Mounting and coverslipping