5ug/ml in H2O for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
Protected from light,Store at -20°C Ships Ice chest + Ice pads Check lot-specific COA for exact specifications.
SDS, COA, datasheet, and spec sheet available for download. Lot-specific COA accessible via lot number lookup.
Cited in 33 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
Hoechst 33258, also known as bis benzimide h 33258 or hoe 33258, is a non embedding bright blfluorescent dye. Dyes have weak fluorescence in solution, and their fluorescence becomes bright after binding with DNA at the minor groove in the DNA poly at sequence rich region in living cells. Therefore, such dyes are also known as DNA probes. Because of the low background, the stained cells do not need washing steps, and the staining is very stable, non-toxic to live cells, and can last for several days or longer after combined with DNA staining. Hoechst 33258 has higher solubility in water than Hoechst 33342, but both dyes have high cell membrane permeability and are widely used for apoptosis detection. After staining, it can be observed by fluorescence microscope or detected by flow cytometry. Take adherent cells (96 well plate) as an example, 100 per well μ L staining solution, 10 ml can be used for the staining of 100 wells.
Product parameters：
Ex/em (bound DNA) = 352/461 nm； Ex/Em (unbound DNA) = 346/460 nm
Matters needing attention：
1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments.
2. if you need to adjust the concentration, please select H598341 and configure the appropriate working fluid concentration by yourself.
3. fluorescent dyes have quenching problems. It is recommended to observe immediately after staining live cells or tissues.
4. for your safety and health, please wear experimental clothes and disposable gloves.
Scope of application：
Nuclear staining
Experimental steps:
1. For fixed cells or tissues
(1) For cell or tissue samples, wash and remove the fixative appropriately after fixation. If immunofluorescence staining is required, first perform immunofluorescence staining, and then follow the subsequent steps to perform Hoechst 33258 staining.
(2) For adherent cells or tissue sections, add a small amount of Hoechst 33258 working solution and cover the sample. For suspended cells, add at least three times the volume of the sample to be tested and mix well. Leave at room temperature for 3-5 minutes.
(3) Remove Hoechst 33258 staining solution and wash 2-3 times with TBST, PBS, or physiological saline for 3-5 minutes each time.
Note: The cleaning steps are optional but not necessary and do not affect dyeing after cleaning.
(4) Observe directly under a fluorescence microscope or observe under a fluorescence microscope after sealing. When a cell undergoes apoptosis, the nucleus of the apoptotic cell can be seen to be densely stained or fragmented and densely stained.
2. For live cells or tissues
(1) Add an appropriate amount of Hoechst 33258 working solution and fully cover the sample to be stained. Typically, 1 mL of staining solution is required per well for a six well plate, and 100 mL is required per well for a 96 well plate μ L's staining solution.
(2) Incubate at room temperature in dark for 10-30 minutes.
(3) Discard the staining solution, wash 2-3 times with PBS, then add 50 μ Perform micrographs using PBS.
Note: The cleaning steps are optional but not necessary and do not affect dyeing after cleaning.
Comprehensive hazard, handling, storage, and regulatory compliance document.
Download SDS →Lot-specific quality data. Enter your lot number to retrieve the exact COA.
Look up COA →Full quality attributes and acceptance criteria for this grade.
View spec sheet →Taxonomy Tree
| Kingdom | Organic compounds |
|---|---|
| Superclass | Organoheterocyclic compounds |
| Class | Benzimidazoles |
| Subclass | Phenylbenzimidazoles |
| Intermediate Tree Nodes | Not available |
| Direct Parent | Phenylbenzimidazoles |
| Alternative Parents | N-arylpiperazines Phenylimidazoles Dialkylarylamines N-methylpiperazines 1-hydroxy-2-unsubstituted benzenoids Benzene and substituted derivatives Heteroaromatic compounds Trialkylamines Azacyclic compounds Organooxygen compounds Hydrochlorides Hydrocarbon derivatives |
| Molecular Framework | Aromatic heteropolycyclic compounds |
| Substituents | Phenylbenzimidazole - N-arylpiperazine - 2-phenylimidazole - Dialkylarylamine - Tertiary aliphatic/aromatic amine - 1-hydroxy-2-unsubstituted benzenoid - Phenol - N-alkylpiperazine - N-methylpiperazine - Monocyclic benzene moiety - 1,4-diazinane - Benzenoid - Piperazine - Azole - Heteroaromatic compound - Imidazole - Tertiary aliphatic amine - Tertiary amine - Azacycle - Hydrochloride - Organooxygen compound - Organonitrogen compound - Hydrocarbon derivative - Organic oxygen compound - Organic nitrogen compound - Amine - Aromatic heteropolycyclic compound |
| Description | This compound belongs to the class of organic compounds known as phenylbenzimidazoles. These are compounds containing a phenylbenzimidazole skeleton, which consists of a benzimidazole moiety where its imidazole ring is attached to a phenyl group. |
| External Descriptors | Not available |
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Find and download the COA for your product by matching the lot number on the packaging.
| Lot Number | Certificate Type | Date | Item |
|---|---|---|---|
 A2420226 | Certificate of Analysis | Jan 16, 2024 | H598341 |
| Melt Point(°C) | 314°C |
|---|---|
| Molecular Weight | 533.900 g/mol |
| XLogP3 | |
| Hydrogen Bond Donor Count | 6 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 3 |
| Exact Mass | 532.131 Da |
| Monoisotopic Mass | 532.131 Da |
| Topological Polar Surface Area | 84.100 Ų |
| Heavy Atom Count | 35 |
| Formal Charge | 0 |
| Complexity | 634.000 |
| Isotope Atom Count | 0 |
| Defined Atom Stereocenter Count | 0 |
| Undefined Atom Stereocenter Count | 0 |
| Defined Bond Stereocenter Count | 0 |
| Undefined Bond Stereocenter Count | 0 |
| The total count of all stereochemical bonds | 0 |
| Covalently-Bonded Unit Count | 4 |
