Hoechst 34580 xHCl(23555-00-2(free base) is a cell-permeable fluorescent dye, is used for staining DNA and nuclei.

Hoechst 34580 is a good candidate for treating Alzheimer’s disease by inhibiting Aβ formation. 50 μM Aβ42 solutions co-incubated with 100, 25, 12.5, 3.125, 0.78, and 0.1, 0.01 μM Hoechst 34580 at 37 °C for 70 h. Hoechst 34580 can inhibit the aggregation of Aβ42 in a dose-dependent manner. The IC50 is obtained by measuring the concentration of Hoechst 34580 while maintaining the Aβ42 concentration which gave 0.86±0.05 μM for Hoechst 34580.

Instructions
I. Solution preparation
1. Stock solution: Dissolve Hoechst 34580 in DMSO to prepare a 1mg/ml stock solution.
Note: The stock solution should be kept away from light and stored at -20°C after aliquoting to avoid repeated freezing and thawing to maintain the activity of the dye.
2. Working solution: Dilute the stock solution to the final concentration (usually 1–10 µg/mL) before the experiment.
Note: It is recommended to use a suitable pure DMEM medium or PBS (without phenol red) for dilution to ensure the best staining effect.
II. Operation steps
1. Cell preparation:
Adherent cells: After culturing cells to an appropriate density, wash 1–2 times with PBS buffer to remove the culture medium residue.
Suspended cells: Collect cells directly, centrifuge and discard the supernatant, and resuspend with PBS.
2. Staining reaction:
1) Resuspend cells or incubate adherent cells with Hoechst 34580 working solution to ensure sufficient mixing.
2) The staining concentration is usually 1–10 µg/mL, and incubate at 37°C in the dark for 10–30 minutes.
3. Washing steps: After staining, wash the cells 2–3 times with PBS buffer to remove unbound dye.
3. Detection and analysis
Fluorescence detection: Use a fluorescence microscope or flow cytometer to detect the fluorescent signal in the cells.
Hoechst 34580 has a high affinity for DNA and can specifically bind to the cell nucleus, showing bright blue fluorescence.
Recommended detection parameters:
Excitation wavelength: 355–360 nm
Emission wavelength: 465–480 nm
4. Data analysis:
Observe and take fluorescent images to analyze the nuclear staining intensity or cell cycle distribution. Positive and negative control groups can be combined for comparison to verify the experimental results.