Thermo Scientific Pierce Hoechst 33342 Fluorescent Stain is a high-quality solution of Hoechst dye for fixed- and live-cell fluorescent staining of DNA and nuclei in cellular imaging techniques.
Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate) is a cell-permeable DNA stain that is excited by ultraviolet light and emits blue fluorescence at 460 to 490nm. Hoechst 33342 binds preferentially to adenine-thymine (A-T) regions of DNA. This stain binds into the minor groove of DNA and exhibits distinct fluorescence emission spectra that are dependent on dye:base pair ratios.
Highlights:
- Hoechst dye – blue fluorescent stain specific for DNA (i.e., nuclei of eukaryotic cells)
- Convenient – provided as an easy-to-use Hoechst dye solution (20mM)
- Cellular imaging – cell-permeable dye is effective for fixed-cell and live-cell staining
- Counterstain – ideal for use alongside detection of specific targets with fluorescent antibodies for fluorescence microscopy or high-content screening (HCS)
Properties of Hoechst Fluorescent Dye:
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Alternative names
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Alternative names
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Hoechst Stain, Hoechst Dye, DNA Content Counterstain
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Chemical name
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2'-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate
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Molecular formula
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C27H28N6O •3HCl •3H2O
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Molecular weight
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615.99
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Excitation wavelength
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346 ±3nm (361nm when bound to dsDNA)
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Emission wavelength
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497 ±3nm
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Extinction coefficient
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Source compound ~47,000/M cm (> 45,000) at 343nm in methanol
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CAS #
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28491-52-3
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Purity
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> 95% (most lots >98%) by HPLC at 240nm
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Solubility
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Product is supplied at 20mM (12.3mg/mL) in water; Hoechst dye is soluble in DMF, water and various non-phosphate aqueous buffers
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Storage
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Store supplied solution at 2 to 8°C protected from light
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Reactive groups
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None; dye binds to minor groove of double-stranded DNA
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Product Details:
As a counterstain in fluorescent imaging, Hoechst dye is compatible with antibodies and other probes labeled with fluorescein and rhodamine dyes, as well as with Thermo Scientific DyLight Fluors. The stable 20mM aqueous stock solution is essentially ready for use.
Hoechst 33342 is used for specifically staining the nuclei of living or fixed cells and tissues. This stain is commonly used in combination with 5-bromo-2'-deoxyuridine (BrdU) labeling to distinguish the compact chromatin of apoptotic nuclei, to identify replicating cells and to sort cells based on their DNA content. Hoechst 33342 and propidium iodide are frequently used together for simultaneous flow cytometric and fluorescence imaging analysis of the stages of apoptosis and cell-cycle distribution.
| Live cell imaging with Hoechst 33342. Live HeLa cells plated on 96-well plates, incubated with Thermo Scientific Hoechst 33342 Solution was added at 1µg/mL for 5 minutes, and imaged with the Thermo Scientific ArrayScan VTI Instrument (Obj. 20X/0.45NA). |
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| Fixed-cell staining with Hoechst dye alongside antibodies. DU145 prostate cancer cells DU145 were grown in 96-well plates, fixed with paraformaldehyde and permeabilized for 15 minutes. Cells were incubated for 1 hour at room temperature with a mouse anti-human HMGB antibody and rabbit anti-human RAGE antibody, then for 45 minutes with orange and green DyLight Fluor goat anti-mouse and goat anti-rabbit secondary antibodies, respectively. Finally, Hoechst 33342 Solution was added at 1µg/mL for 5 minutes. Stained cells were imaged with the Thermo Scientific ArrayScan VTI Instrument (Obj. 20X/0.45NA). |
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| Fixed-tissue staining with Hoechst dye and an antibody. Formalin-fixed paraffin embedded tonsil control tissue was deparaffinized, subjected to heat-induced epitope retrieval using citrate buffer, and then blocked. The sample was incubated for 30 minutes at room temperature with mouse anti-vimentin antibody, then for 30 minutes with orange DyLight Fluor goat anti-mouse secondary antibody. Finally, Hoechst 33342 Solution was added at 1µg/mL for 5 minutes. The stained tissue was imaged with the Zeiss Axio Observer .Z1 Microscope (Obj. 20X/0.4NA). |
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| Cellular imaging with Hoechst 33342. A549 human lung cancer cells were grown on 96-well plates, fixed with paraformaldehyde and permeabilized for 15 minutes. Cells were incubated for 30 minutes at room temperature with mouse anti-human alpha-tubulin antibody, then for 45 minutes with orange DyLight Fluor goat anti-mouse secondary antibody. Finally, Hoechst 33342 Solution was added at 1µg/mL for 5 minutes. Stained cells were imaged with the Thermo Scientific ArrayScan VTI Instrument (Obj. 20X/0.45NA). |
Related Products:
DyLight Reactive Fluors
All Fluorescent Labeling Reagents – including AMCA, FITC, and TRITC
Immunohistochemistry Reagents – including ABC and DAB staining kits
Thermo Scientific High-Content Screening (HCS) Reagents
Related Resources:
Review of Immunohistochemistry (IHC) Methods
Review of IHC Counterstain Methods
Tech Tip #31 – Calculate dye:protein (F/P) molar ratios
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