Hoechst 33258 analog 3 is part of a family of blue fluorescent dyes used to stain DNA.

The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra. Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescent light around an emission maximum at 461 nm. Unbound dye has its maximum fluorescence emission in the 510-540 nm range. Hoechst dyes are soluble in water and in organic solvents such as dimethylformamide or dimethyl sulfoxide. Concentrations can be achieved of up to 10 mg/mL. Aqueous solutions are stable at 2-6 °C for at least six months when protected from light. For long-term storage, the solutions are instead frozen at ≤-20 °C. Although the dyes can bind to all nucleic acids, AT-rich double-stranded DNA strands enhance fluorescence considerably. Hoechst dyes are cell-permeable and can bind to DNA in live or fixed cells.

Instructions
1. Dissolution and dilution:
Hoechst 33258 Analog 3 is usually dissolved in DMSO (if applicable) or PBS. Before use, the dye can be diluted according to the concentration recommended by the manufacturer.
The generally recommended staining concentration is 10ug/ml, and the specific concentration needs to be optimized according to the experimental requirements.
2. Staining steps:
a. Fixation: If cells need to be fixed (for example, with formaldehyde or ice-cold methanol), fix them first.
b. Staining: Add an appropriate amount of Hoechst 33258 Analog 3 solution to the cell culture plate and stain for about 15-30 minutes at room temperature.
c. Rinse: Gently wash the cells with PBS or an appropriate buffer to remove unbound dye.
3. Fluorescence microscopy: The stained cells can be observed under ultraviolet light.
Hoechst 33258 Analog 3 emits blue fluorescence, usually with an excitation light of 360-380 nm and an emission light in the range of 450-470 nm.