Bisbenzimide (Hoechst 33258) is a fluorescent DNA and chromosome stain that preferentially binds AT-rich regions of double-stranded DNA. DNA binding increases fluorescence, producing blue emission that supports visualization of nuclei and chromosomes by fluorescence microscopy and related fluorescence-based methods.
Synonyms: Hoechst 33258; bisbenzimide H 33258; HOE 33258.
Hoechst 33258 associates preferentially with the minor groove of AT-rich DNA sequences. This sequence-dependent interaction makes the dye useful for nuclear and chromosome staining as well as fluorescence measurements involving DNA content or base composition.
DNA-bound Hoechst 33258 typically exhibits ultraviolet excitation near 355 nm and blue fluorescence near 465 nm. Exact spectral behavior and fluorescence intensity depend on DNA binding, buffer conditions, dye concentration, and the optical configuration used for measurement.
Key Properties
- Fluorescent bisbenzimide DNA and chromosome stain
- Preferential binding to AT-rich regions of double-stranded DNA
- CAS number: 23491-45-4
- Purity (HPLC): >98.0%
- Appearance: yellow-green solid
- Water-soluble
- Available as 100 mg; catalog 09460
Applications
Fluorescent Nuclear & Chromosome Staining
Hoechst 33258 is used to fluorescently label DNA in nuclei and chromosomes for visualization by fluorescence microscopy. Staining time should be adjusted according to specimen type, section thickness, dye concentration, and imaging conditions.
DNA Visualization
Binding to double-stranded DNA enhances Hoechst 33258 fluorescence, allowing DNA-rich structures to be visualized with ultraviolet excitation and blue-emission detection.
DNA Fluorometry
Hoechst 33258 has been used in fluorescence-based DNA measurements where changes in dye fluorescence provide information about DNA content. Quantitative methods require appropriate DNA standards and consistent assay conditions.
DNA Base-Composition Research
Because fluorescence is influenced by DNA base composition and Hoechst 33258 preferentially associates with AT-rich sequences, the dye has also been used in studies examining DNA composition and sequence-dependent fluorescence behavior.
Compatibility & Bonding Considerations
- Preferentially associates with the minor groove of AT-rich double-stranded DNA
- DNA-bound dye typically exhibits UV excitation and blue fluorescence near 355/465 nm
- Compatible with fluorescence instrumentation configured for UV excitation and blue-emission detection
- Fluorescence intensity depends on DNA concentration, base composition, staining conditions, and measurement settings
Staining intensity and fluorescence measurements can vary with dye concentration, section thickness, DNA accessibility, buffer composition, pH, incubation time, and imaging parameters. Keep experimental and acquisition conditions consistent when comparing samples.
Application Tips
- Adjust staining time according to specimen type and section thickness; a 2–10 minute interval may be used as a starting range for appropriate staining procedures
- Maintain consistent dye concentration and incubation conditions when comparing samples
- Use an appropriate UV-excitation and blue-emission filter configuration for fluorescence imaging
- Minimize unnecessary light exposure during preparation and imaging to reduce changes in fluorescence signal
- For quantitative fluorescence measurements, use suitable DNA standards and consistent instrument settings
Handling & Storage
- Store at 4°C in a cool, well-ventilated location
- Keep the container tightly closed when not in use
- Handle with good laboratory ventilation and avoid breathing dust
- Wear protective gloves, safety glasses, and suitable protective clothing
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FAQ
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1. What is Hoechst 33258 used for?
Hoechst 33258 is a fluorescent DNA stain used for visualization of nuclei and chromosomes and in fluorescence-based studies involving DNA content or base composition.
2. Are bisbenzimide and Hoechst 33258 the same?
Yes. For catalog 09460, Bisbenzimide refers to Hoechst 33258, a bisbenzimide fluorescent DNA-binding dye. The CAS number is 23491-45-4.
3. How does Hoechst 33258 bind DNA?
Hoechst 33258 preferentially binds in the minor groove of AT-rich regions of double-stranded DNA. DNA binding increases the fluorescence of the dye and produces the blue nuclear and chromosome signal used in fluorescence imaging.
4. What are the excitation and emission wavelengths of Hoechst 33258?
DNA-bound Hoechst 33258 typically has an excitation maximum near 355 nm and an emission maximum near 465 nm. Exact spectral values can vary with DNA binding and solution conditions.
5. Is Hoechst 33258 the same as Hoechst 33342?
No. Hoechst 33258 and Hoechst 33342 are structurally related bisbenzimide DNA stains, but they are distinct compounds and should not be treated as interchangeable reagents. Select the dye specified by the validated staining or imaging protocol.
Safety & Documentation
Hoechst 33258 is harmful if swallowed or inhaled and can cause skin irritation. Use appropriate laboratory ventilation, protective gloves, eye protection, and suitable protective clothing. Review the Safety Data Sheet (SDS) for complete handling and safety information and the product specification for current quality-control requirements. For quantities larger than the listed 100 mg package, request a bulk quote.
