Hoechst 34580 serves as a blue-emitting fluorescent dye that selectively binds to adenine-thymine-rich regions in double-stranded DNA. It exhibits excitation/emission peaks at 380/438 nm when bound to DNA, with fluorescence intensity sensitive to solvent pH. Unbound dye emits green light in the 510–540 nm range, more pronounced at higher concentrations or inadequate washing. While capable of penetrating living cells, it is less permeable than Hoechst 33342. Importantly, the dye is less toxic than DAPI, ensuring better cell viability post-staining.
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