Hoechst 33258, also known as bisBenzimide H 33258 or HOE 33258, is a cell-permeable blue fluorescent dye with relatively low cytotoxicity. Hoechst 33258 staining is commonly used in apoptosis detection, followed by observation under a fluorescence microscope or detection via flow cytometry. Hoechst 33258 is also frequently used for routine cell nuclei staining or general DNA staining. The maximum excitation wavelength of Hoechst 33258 is 346 nm, and the maximum emission wavelength is 460 nm; when bound to double-stranded DNA, the maximum excitation wavelength shifts to 352 nm and the maximum emission wavelength to 461 nm. This Hoechst 33258 staining solution can be directly used for nucleus staining of fixed cells or tissues, as well as for live cells or tissues.

Usage Instructions:
Dilute the Hoechst 33258 staining solution 100-fold with saline or PBS before use to prepare the working solution.

1. For fixed cells or tissues:
a. For cell or tissue samples, fix and then wash appropriately to remove the fixative. If immunofluorescence staining is required, perform it first, followed by Hoechst 33258 staining as described below. If no additional staining is needed, proceed directly to Hoechst 33258 staining.
b. For adherent cells or tissue sections, add a small amount of Hoechst 33258 working solution sufficient to cover the sample; for suspended cells, add at least three volumes of working solution relative to the sample volume, and mix well. Incubate at room temperature for 3–5 minutes.
c. Remove the Hoechst 33258 staining solution and wash 2–3 times with TBST, PBS, or saline, 3–5 minutes each wash.
d. Observe directly under a fluorescence microscope or after mounting. In apoptotic cells, the nuclei will appear highly condensed or fragmented with intense staining.

2. For live cells or tissues:
a. Add an appropriate amount of Hoechst 33258 working solution, ensuring complete coverage of the sample. Typically, add 1 mL of working solution per well of a 6-well plate, or 100 μL per well of a 96-well plate.
b. Incubate at a temperature suitable for cell culture for 20–30 minutes. Discard the staining solution and wash 2–3 times with PBS or culture medium before fluorescence detection.

Precautions:
All fluorescent dyes are prone to quenching. To slow down fluorescence quenching, an anti-fade mounting medium can be used. It is recommended to complete detection on the same day after staining; live cells or tissues should be observed immediately after staining.
For your safety and health, please wear a lab coat and disposable gloves during handling.