DAPI and Hoechst specifically stain double-stranded DNA. They have very similar spectral property (Ex: UV, Em: blue/cyan) and can be used interchangeably in many instances. However, a major difference is that DAPI is cell impermeant and Hoechst is cell-permeant. Hoechst precipitates in phosphate buffer when the concentration is high. However, this is not an issue in regular working condition.

To perform DAPI or Hoechst staining, simply dilute stock solution 1:1000 in the medium and incubate the sample in working solution for at least 30 minutes. We often wash the sample with buffered saline several times before mounting for observation, although wash is not necessary. For best results, DAPI/Hoechst stained sample should be mounted in buffered glycerol.

To make stock,

dissolve Hoechst 33342 (Molecular Probes H1399; Sigma B2261) in ddH₂O at 10 mg/mL, or

DAPI (Molecular Probes D1306; Sigma D9542) in ddH₂O or DMF at 5 mg/mL.

Note both Hoechst and DAPI are potential carcinogens. Handle with care.

Other substitutes for nucleic acid counterstaining are

propidium iodide (Sigma P4170; Em: red)

Sytox Green (Molecular Probes S7020; Em: green)

7-aminoactinomycin D (7-AAD; Molecular Probes A1310; Em: red)