Hoechst 33342 alkyne is an excellent building block for developing site-specific nucleic acid probes via the well-known click chemistry, i.e., Copper-Catalyzed Azide–Alkyne Cycloaddition (CuAAC). It can be readily conjugated to azido-containing biomolecules (such as azido-modified antibodies, peptide azides and azido-modified oligos). Hoechst 33342 is a DNA-specific dye that binds to the minor groove of double-stranded DNA. Its ability to readily penetrate live cell membranes makes it suitable for staining live cells. This property allows researchers to monitor nuclear morphology and DNA content in living cells. Hoechst 33342 is commonly used in fluorescence microscopy to visualize the nucleus of cells. It selectively stains the DNA in the cell nucleus and is often used in combination with other fluorochromes to study cell structure and dynamics. In addition, Hoechst 33342 has also been used in the identification and isolation of stem cells based on their ability to efflux the dye. This property is exploited in a technique called side population analysis, commonly used in stem cell research.