Hoechst likes to play hide and seek … use it with caution!

Abstract We describe an unexpected feature of the dye Hoechst 33342 that may lead to misinterpretation of fluorescence assay results. When dissolved in aqueous solvent, Hoechst 33342 massively adsorbs on the polytetrafluoroethylene (PTFE)-coated stirrer and on the quartz cuvette. The interaction between the dye and the PTFE is stronger than that between the dye and the quartz. We show that Hoechst 33342 adsorption on quartz is due to its amphiphilic properties, as it depends on the buffer ionic strength and on pH. We suggest a procedure to detect and remove any residual Hoechst 33342.