Hoechst stain

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Transmission image of HeLa cells, with overlay of Hoechst 33258 staining (blue). The leftmost cell is in the metaphase stage of mitosis; its chromosomes fluoresce brightly because they contain highly compacted DNA.

Hoechst (magenta) bound to the minor groove of DNA (green and blue). From PDB 264D.

The Hoechst stains are part of a family of fluorescent stains for labelling DNA in fluorescence microscopy. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. Two of these closely related bis-benzimides are commonly used: Hoechst 33258 and Hoechst 33342.

Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescence light around an emission maximum at 461 nm. The Hoechst stains may be used on live or fixed cells, and are often used as a substitute for another nucleic acid stain, DAPI. The key difference between them is that the additional ethyl group of Hoechst 33342 renders it more lipophilic, and thus more able to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant.

These dyes can also be used to detect the contents of a sample DNA by plotting a standard emission-to-content curve.

Because the Hoechst stains bind to DNA, they can disrupt DNA replication during cell division. Consequently they are potentially mutagenic and carcinogenic. Care should be taken in their handling and disposal.