Hoechst 33342 trihydrochloride (bisBenzimide H 33342 trihydrochloride;HOE 33342 trihydrochloride) is a membrane permeant DNA stain with blue fluorescence.

Instructions
I. Solution preparation
1. Stock solution: Dissolve Hoechst 33342 trihydrochloride in deionized water or an appropriate solvent (such as PBS), usually prepared as a 1 mg/mL stock solution.
2. Working solution: Dilute the stock solution to a working concentration, usually 10 µg/mL, as required by the experiment. The specific concentration can be optimized according to the experimental needs.
II. Operation steps
1. Cell culture: Inoculate cells into appropriate culture dishes until the appropriate density is reached.
2. Staining: Add Hoechst 33342 working solution to the culture medium and incubate for 10–30 minutes, usually at 37°C. For live cell staining, the time can be adjusted according to experimental needs.
3. Washing: Wash the cells with PBS to remove unbound dye.
4. Imaging: Observe the cells using a fluorescence microscope. Hoechst 33342 emits blue fluorescence (455 nm) when excited by ultraviolet light (360–365 nm).
5. Application areas:
1) Cell cycle analysis: Hoechst 33342 can be used to label DNA in the cell nucleus, bind to DNA, and can be used for flow cytometry (FACS) analysis of the cell cycle.
2) Chromosome research: It is often used as a chromosome stain to help observe chromosomes in cell division.
3) Live cell imaging: Due to its membrane permeability, it can be used for imaging of live cells, providing clear visualization of the cell nucleus.
Precautions:
1. Photosensitivity: Hoechst 33342 is sensitive to light and should be protected from direct exposure to strong light and stored in dark places.
2. Toxicity: Although the dye is suitable for staining live cells, high concentrations or long-term exposure may have toxic effects on cells, so the dye concentration and staining time should be controlled when used.
3. Solubility: Ensure that the stock solution and working solution are completely dissolved before use to avoid the formation of precipitation.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

H2O: 5.67 mg/mL (10.09 mM), Sonication is recommended.

DMSO: 46 mg/mL (81.86 mM), Sonication is recommended.