Amyloid-β[1]
Hoechst 34580 tetrahydrochloride targets the minor groove of double-stranded DNA, with a strong preference for AT-rich regions. Binding to DNA markedly enhances fluorescence. The compound is a live cell nuclear labeling dye that binds nucleic acids via binding to the minor groove in the DNA double strand. It is also categorized as an Amyloid-β inhibitor in some reference sources.

Hoechst working solution preparation 1.1 Hoechst stock solution preparation Make a 1 mg/mL Hoechst stock solution using DMSO. Note: After aliquots, Hoechst storage solution should be kept at -4°C or -20°C out of direct sunlight. 1.2 Making the working solution: Use either PBS-diluted storage solution or heated serum-free cell culture medium, bringing the final concentration of the Hoechst working solution to 10 μg/mL. Note: Before using, please make sure that the Hoechst working fluid concentration is appropriate for the current circumstances. 2. Suspended cell staining (2.1): Centrifuge cells, add PBS, then wash twice for five minutes each time. The density of cells is 1×106/mL. 2.2 After adding 1 mL of Hoechst working solution, incubate for 3–10 minutes at room temperature. 2.3 Centrifuge for 3–4 minutes at 400 g, then remove the supernatant. 2.4 After adding PBS, wash the cells twice for five minutes each. 2.5 Re-suspend the cells in 1 milliliter of PBS or serum-free media, and use a flow cytometer or fluorescence microscope to observe. 3. Adherent cell staining (3.1): Grow adherent cells on sterile coverslips. 3.2 Aspirate extra culture medium after removing the coverslip from the medium. 3.3 Add 100 μL of the dye working solution, give the cells a full shake to cover them, and then let them sit for three to ten minutes. 3.4 Use a flow cytometer or fluorescence microscope to observe after aspirating the dye working solution and washing it twice in culture media for a total of five minutes each time. Observations 1. Kindly modify the Hoechst working fluid concentration based on the current circumstances. 2. This product may not be used for clinical diagnosis or treatment, nor may it be included into food or medication. It is intended solely for professional use in scientific study. 3. Please wear disposable gloves and a lab coat for your health and safety.

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In vitro, Hoechst 34580 tetrahydrochloride functions as a nuclear marker dye that selectively targets A/T-rich double-stranded DNA. The compound's fluorescence is significantly enhanced upon binding to DNA, making it valuable for live cell labeling applications. It enables nuclear staining and DNA visualization in various cell-based assays. The dye is commonly used in fluorescence microscopy, imaging, and flow cytometry for nuclear counterstaining.

In vivo, Hoechst 34580 tetrahydrochloride has been studied in the context of amyloid-β research, where it is categorized as an Amyloid-β inhibitor. As a DNA-binding dye, its primary in vivo applications are in imaging and labeling studies rather than therapeutic use. The compound's ability to bind AT-rich regions of DNA makes it useful for studying chromatin structure and nuclear morphology in living organisms. However, comprehensive in vivo pharmacological studies are limited.

For non-cell-based receptor binding assays, Hoechst 34580 tetrahydrochloride can be evaluated using purified DNA or chromatin preparations. DNA binding assays are performed by incubating increasing concentrations of the compound with double-stranded DNA in appropriate buffer systems. Fluorescence enhancement upon DNA binding is measured using a fluorescence spectrophotometer with excitation at approximately 392 nm and emission at 440 nm. Competitive binding experiments can be performed using unlabeled DNA or other DNA-binding compounds. Binding affinity is calculated from fluorescence titration curves using nonlinear regression analysis.

For in vitro cellular assays, cells are cultured in appropriate media and stained with Hoechst 34580 tetrahydrochloride. For live cell labeling, cells are incubated with the dye at appropriate concentrations for 15-30 minutes at 37°C. For fixed cell staining, cells are fixed with paraformaldehyde or methanol prior to dye incubation. Stained cells are washed and examined using fluorescence microscopy with a DAPI or Hoechst filter set (excitation ~350-400 nm, emission ~420-460 nm). Nuclear morphology and DNA content can be assessed using image analysis software. For flow cytometry, stained cells are analyzed using a UV laser for DNA content analysis and cell cycle studies.

For in vivo animal studies, Hoechst 34580 tetrahydrochloride can be administered to animals via intravenous injection for imaging applications. In tumor models, the dye may be used to label tumor cell nuclei for assessing tumor burden or treatment response. In developmental biology studies, the compound can be used to visualize nuclear structures in whole-mount embryos. Dosing and administration routes vary depending on the specific imaging application and target tissue. Tissue samples are collected and examined using fluorescence microscopy.

Hoechst 34580 tetrahydrochloride is a cell-permeable dye with improved aqueous solubility and stability due to its tetrahydrochloride salt form. The compound has a molecular weight of 597.41 and a molecular formula of C27H33Cl4N7. It is supplied in high purity (≥95%). The compound should be stored at -20°C for long-term stability. Its fluorescence properties (excitation/emission ~392/440 nm when DNA-bound) make it compatible with standard DAPI filter sets.

The toxicity profile of Hoechst 34580 tetrahydrochloride has not been extensively reported in the available literature. As a DNA-binding fluorescent dye, potential toxicities may include genotoxicity at high concentrations due to DNA intercalation or minor groove binding. The compound is for research use only and not for human consumption. Standard safety precautions should be observed when handling the compound, including appropriate personal protective equipment. The dye is generally considered safe for standard cell labeling applications at recommended concentrations.

[1]. Discovery of DNA dyes Hoechst 34580 and 33342 as good candidates for inhibiting amyloid beta formation: in silico and in vitro study. J Comput Aided Mol Des. 2016 Aug;30(8):639-50.

Hoechst 34580 tetrahydrochloride (HOE 34580 tetrahydrochloride) is a cell-permeable bisbenzimidazole dye used for bright blue fluorescent staining of DNA and nuclei in live or fixed cells. It selectively binds AT-rich regions of DNA and emits fluorescence upon binding, enabling nuclear staining and DNA visualization. The compound has a molecular weight of 597.41 and is supplied as a tetrahydrochloride salt for improved solubility. It is used in fluorescence microscopy, imaging, flow cytometry, and as a nuclear counterstain.

597.152

2310135-08-9

Hoechst 34580;23555-00-2

131704503

Light yellow to khaki solid powder

6

5

4

38

672

0

CN1CCN(CC1)C2=CC3=C(C=C2)N=C(N3)C4=CC5=C(C=C4)N=C(N5)C6=CC=C(C=C6)N(C)C.Cl.Cl.Cl.Cl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 2.67 mg/mL (4.47 mM)

Solubility in Formulation 1: 6.25 mg/mL (10.46 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)