The Click-iT™ EdU Colorimetric IHC (Immunohistochemistry) Detection Kit is a superior alternative to traditional methods for the colorimetric detection of proliferating cells within tissue sections. In this assay, 5-ethynyl-2'-deoxyuridine (EdU), a nucleoside analog of thymidine containing an alkyne moiety, is incorporated into newly synthesized DNA. When biotin containing an azide group is added, a brief 'click reaction' attaches the azide to the alkyne moiety on the EdU molecule. Upon addition of streptavidin–peroxidase and a peroxidase substrate, an enzymatic reaction generates a signal that can be detected with light microscopy and stored for future analysis.

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• Optimized—enables colorimetric detection of proliferating cells in either tissue or cell samples
• Efficient—no denaturation steps or harsh treatment required
• Robust—improved preservation of cell morphology and the cellular environment offers content-rich results
• Consistent—not dependent on variable antibody lots for detection
• Simple—designed to work consistently, in less time than traditional methods

Traditional methods yield inconsistent results
Measuring changes to cell proliferation is a fundamental method for assessing cell health, determining genotoxicity, and evaluating anti-cancer drug research. The most accurate method of doing this is by directly measuring DNA synthesis. The traditional method utilizes the nucleoside analog 5-bromo-2'-deoxyuridine (BrdU), a nucleoside analog of thymidine, which is incorporated into newly transcribed DNA. After incorporation, the samples are treated with harsh methods (HCl, heat, or enzymes) to denature the DNA and expose the BrdU molecules to detection by anti-BrdU antibodies. These harsh treatments can adversely affect sample integrity, cell morphology, and image quality. In addition, manufacturing variability, nonspecific binding, and cross-reactivity issues can result in inconsistent results.

Advantages over other assays
The Click-iT EdU Colorimetric IHC Detection Kit offers many advantages over traditional methods like the BrdU assay. The small size of the biotin-azide moiety allows efficient detection of incorporated EdU using mild conditions. In contrast, BrdU assays require harsh denaturation methods using HCl and/or heat-induced epitope retrieval (HIER). Additionally, unlike the BrdU assay, which relies on antibodies that can sometimes exhibit nonspecific binding, the Click-iT EdU Colorimetric IHC Detection Kit utilizes a bioorthogonal (biologically unique) moiety—resulting in low background signal, high detection sensitivities, and no cross-reactivity issues.

The kit contains all the components needed to detect incorporated EdU present in formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit includes sufficient reagents for labeling fifty (50) 18 x 18 mm coverslips using 100 μL reaction reagent per test.