Abstract
Many peroxidase inhibitors have been used in horseradish peroxidase (HRP) mediated immunostaining and in situ hybridization to quench background peroxidase activity. However, the efficacy of these inhibitors has been controversial, partially due to the lack of a quantitative study. Tyramide signal amplification (TSA) is much more sensitive than other HRP-mediated methods but its super-sensitivity also demands effective inhibition of background peroxidase activity. In searching for an effective peroxidase inhibitor, we have systematically evaluated the efficacy of several peroxidase inhibitors by quantifying the fluorescence intensity in cultured fibroblasts and tissue sections treated with the inhibitors. For cultured cells, 0.05 mM of phenylhydrazine and 1 unit/ml of glucose oxidase gave only moderate inhibition of HRP activity while 1 mM of sodium azide (NaN3), 3% of hydrogen peroxide (H2O2), NaN3/H2O2 combined and 0.02 N hydrochloric acid (HCl) provided more complete inhibition. However, the inhibitory effect of NaN3/H2O2 is reversible upon removal of the inhibitors and followed by incubation and wash to mimic antibody interactions. Similar results were obtained from rat skin wound tissues that have strong endogenous peroxidase activity. Our results recommend the use of HCl and caution the use of phenylhydrazine, glucose oxidase, NaN3 and H2O2 as potent peroxidase inhibitors.
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Acknowledgements
Dr Livingston Van De Water and Ms. Jessica Germond for help in tissue preparation. This research was supported by NIH grants R01-GM70560 to GL, R25-GM062460 to SA and T32-HL07194 to LAM.
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Liu, G., Amin, S., Okuhama, N.N. et al. A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry. Histochem Cell Biol 126, 283–291 (2006). https://doi.org/10.1007/s00418-006-0161-x
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DOI: https://doi.org/10.1007/s00418-006-0161-x