How does Hoechst work?
Posted May 7, 2021
Hoechst dyes are popular blue fluorescent, nuclear-specific dyes used to stain live or fixed cells. They bind preferentially to adenine-thymine (A-T) regions of DNA and exhibit distinct fluorescence emission spectra dependent on dye:base pair ratios. Hoechst dyes are excited by UV light (~360 nm) or mercury-arc lamps, or UV lasers and emit a broad spectrum of blue light with a maximum in the 460 nm region. The dyes have minimal fluorescence in solution, but their fluorescence increases ~30-fold upon DNA binding, which ensures a good signal-to-noise ratio. Therefore, they can be used to stain cells without a wash step. The staining is very stable and non-toxic.
Hoechst 33342 and Hoechst 33258 are structurally similar dyes that perform comparably as nuclear counterstains. Hoechst 33528 is slightly more water soluble than Hoechst 33342, but both dyes are highly cell membrane-permeant and widely used in cell cycle studies and as nuclear counterstains for live or fixed cells. They are typically used for staining at 1 ug/mL.