Hoechst 33342 Protocol for Imaging | Thermo Fisher Scientific

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:

Cells growing in culture
Hoechst 33342, trihydrochloride, trihydrate (Cat. No. H1399)
Phosphate-buffered saline (PBS)
Fluorescence microscope

Protocol

Preparing Hoechst dye stock solution

1. 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

1. Culture cells in an appropriate medium and vessel for fluorescence microscopy.

2. Prepare the Hoechst staining solution by diluting the Hoechst stock solution 1:2,000 in PBS.

3. Remove the medium.

4. Add sufficient staining solution to cover the cells.

5. Incubate for 5–10 minutes, protected from light.

6. Optional: You may image directly in the staining solution, if you wish.

7. Remove the staining solution.

8. Wash the cells 3 times in PBS.

9. Image the cells.

Spectral information and storage

	Hoechst 33342
Excitation/Emission (nm)	350/461
Standard filter set	DAPI
EVOS Light Cube	DAPI
Storage conditions	2–6°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. 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.