Image of Hoechst® 33342 packaging

Nuclear counterstain for fluorescence microscopy

Invitrogen Hoechst 33342 nucleic acid stain is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA. This dye is often used to distinguish condensed pycnotic nuclei in apoptotic cells and for cell cycle studies in combination with BrdU. It is also available as a solution (Cat. No. H3570).

This protocol can be used for:

  • Nucleic acid (nuclear) staining in fluorescence microscopy

This protocol should not be used for:

  • Flow cytometry

You will need the following for this protocol:

Simulate your staining experiment before you run it

The Stain-iT simulator can help reduce trial and error and wasted reagents.

Launch simulator

Protocol

Preparing Hoechst dye stock solution

Hoechst stock solution preparation1. Prepare the Hoechst dye stock solution by dissolving the contents of one vial (100 mg) in 10 mL of deionized water (diH2O) to create a 10 mg/mL (16.23 mM) solution. Note: Hoechst dye has poor solubility in water, so sonicate as necessary to dissolve. The 10 mg/mL Hoechst stock solution may be stored at 2–6°C for up to 6 months or at ≤–20°C for longer periods.

Labeling cells

Cell culture in dish1. Culture cells in an appropriate medium and vessel for fluorescence microscopy.
Prepare Hoechst staining solution2. Prepare the Hoechst staining solution by diluting the Hoechst stock solution 1:2,000 in PBS.
Remove medium from cells3. Remove the medium.
Add staining solution4. Add sufficient staining solution to cover the cells.
Incubate 5–10 minutes5. Incubate for 5–10 minutes, protected from light.
Optional direct imaging6. Optional: You may image directly in the staining solution, if you wish.
Remove staining solution7. Remove the staining solution.
Wash cells in PBS8. Wash the cells 3 times in PBS.
Image cells9. Image the cells.
Spectral information and storage
 Hoechst 33342
Excitation/Emission (nm)350/461
Standard filter setDAPI
EVOS Light CubeDAPI
Storage conditions2–6°C or ≤–20°C

 

 

Protocol tips

  • Hoechst dye is a known mutagen and should be handled with care.
  • Dissolving Hoechst dye in PBS is not recommended, but phosphate-containing buffers may be used with dilute solutions of the dye.
  • Unbound Hoechst dye has a maximum emission in the 510–540 nm range; a green haze may be observed if too much dye is applied.
  • The fluorescence signal from Hoechst dye is quenched by BrdU.
Multicolor staining of human fibroblasts
Multicolor staining of human fibroblasts. Cultured human neonatal dermal fibroblasts were fixed and permeabilized, with staining for tubulin (red fluorescence), actin (green fluorescence), and nuclei (blue-fluorescent Hoechst 33342 stain.