A Hybrid Detection Method Based on Peroxidase-mediated Signal Amplification and Click Chemistry for Highly Sensitive Background-free Immunofluorescent Staining

• Published in: The journal of histochemistry and cytochemistry Vol. 67; no. 10; pp. 771 - 782
• Main Authors: Antonov, Stanislav A., Novosadova, Ekaterina V., Kobylansky, Andrey G., Tarantul, Vyacheslav Z., Grivennikov, Igor A.
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA SAGE Publications 01.10.2019
• Subjects: copper-catalyzed azide-alkyne cycloaddition; catalyzed reporter deposition; immunofluorescence; tyramide signal amplification
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1369/0022155419864113

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is increasingly used for detection of various macromolecules and metabolites in biological samples. Here, we present a detailed analysis of the CuAAC reaction conditions in cells and tissue sections. Using the optimized CuAAC conditions, we have devised a highly sensitive immunostaining technique, based on the tyramide signal amplification/catalyzed reporter deposition (TSA/CARD) method with a novel alkyne tyramide substrate. The described method offers improved detection threshold compared to conventional immunofluorescent staining and produces significantly lower non-specific background than TSA/CARD with fluorescent tyramides.