| Product Description | 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. |
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A2420226
