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Hoechst 33342 Fluorescent Stain


A blue nuclear counterstain for fluorescence microscopy and cellular imaging.

Pierce Hoechst 33342 Fluorescent Stain

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:

Structure Chemical structure of Hoechst 33342 fluorescent dye
Alternative names Hoechst Stain, Hoechst Dye, DNA Content Counterstain
Chemical name 2'-(4-Ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate
Molecular formula C27H28N6O •3HCl •3H2O
Molecular weight 615.99
Excitation wavelength 346 ±3nm (361nm when bound to dsDNA)
Emission wavelength 497 ±3nm
Extinction coefficient Source compound ~47,000/M cm (> 45,000) at 343nm in methanol
CAS # 28491-52-3
Purity > 95% (most lots >98%) by HPLC at 240nm
Solubility Product is supplied at 20mM (12.3mg/mL) in water; Hoechst dye is soluble in DMF, water and various non-phosphate aqueous buffers
Storage Store supplied solution at 2 to 8°C protected from light
Reactive groups None; dye binds to minor groove of double-stranded DNA

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). Live cell imaging with Hoechst Dye

Cellular imaging with DAPI and phalloidin probes.
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).

Cellular imaging with DAPI and phalloidin probes.
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).

Cellular imaging with DAPI and phalloidin probes.
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 Resources:

Immunohistochemistry (IHC) technical guide
IHC counterstains technical guide

Related Products:

DyLight Reactive Fluors
All Fluorescent Labeling Reagents – including AMCA, FITC, and TRITC
Immunohistochemistry Reagents – including ABC and DAB staining kits
Cellular Imaging and Analysis Reagents (formerly Cellomics Products)


Ordering Information
 
Product # Description Pkg. Size Instructions MSDS CofA Price
62249 Hoechst 33342 Solution
Formulation: Aqueous solution of 12.3mg/mL (20mM) Hoechst 33342 (MW 615.99; CAS 23491-52-3) in amber glass vial
Sufficient For: At least 60L of typical 1X (1μg/mL) working solution
5mL Product Instructions for product #62249 Hoechst 33342 Solution MSDS for product #62249 Hoechst 33342 Solution $85.00

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Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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