Hoechst 33258 is also known as bisBenzimide H 33258 or HOE 33258, with the molecular formula of C₂₅H₂₄N₆O·3HCl, a molecular weight of 533.88 and a CAS Number of 23491-45-4. It is a blue fluorescent dye capable of penetrating cell membranes with low cytotoxicity, and is commonly used for the detection of cell apoptosis. After staining, observation is performed under a fluorescence microscope or detection via a flow cytometer. Hoechst 33258 is also applied for general nuclear staining and DNA staining. The maximum excitation wavelength of Hoechst 33258 is 346 nm, and its maximum emission wavelength is 460 nm. Upon binding to double-stranded DNA, its maximum excitation wavelength shifts to 352 nm and the maximum emission wavelength to 461 nm.
Antifade Mounting Medium (Containing Hoechst 33258), also referred to as Anti-fade Fluorescent Mountant with Hoechst 33258, can be directly used for nuclear staining of fixed cells or tissues, as well as for live cells or tissues without additional mounting operations. This reagent is for research use only and not intended for clinical diagnosis or any other applications.
Materials to be Prepared by the User:
1. Fluorescence microscope, micropipette
2. Distilled water, PBS or normal saline
Operating Procedures (For Reference Only):
(I) Staining of Fixed Tissue Cells
1. For cell or tissue samples, rinse to remove the fixative after fixation. If immunofluorescence staining is required, perform it first, then conduct Hoechst 33258 staining according to the subsequent steps upon completion of immunofluorescence staining; if no other staining is needed, proceed directly to the subsequent Hoechst 33258 staining. For adherent cells or tissue sections, add a small amount of Anti-fade Fluorescent Mounting Medium (with Hoechst 33258) to just cover the samples. For suspension cells, add at least 3 times the volume of the sample to be stained of Anti-fade Fluorescent Mounting Medium (with Hoechst 33258) and mix thoroughly.
2. Incubate at room temperature for 5–8 minutes.
3. Gently aspirate the Anti-fade Fluorescent Mounting Medium (with Hoechst 33258).
4. Wash 2–3 times with sterile PBS or normal saline for 3–5 minutes each time.
5. Examine directly under a fluorescence microscope without mounting.
(II) Live Cell Staining
1. Culture cells in 96-well, 24-well or 6-well plates to the appropriate state. Add the suitable volume of Anti-fade Fluorescent Mounting Medium (with Hoechst 33258) at the ratio of 100 μl for 96-well plates, 500 μl for 24-well plates and 1 ml for 6-well plates to fully cover the cells.
2. Incubate for 20–30 minutes under optimal cell culture conditions.
3. Gently aspirate the Anti-fade Fluorescent Mounting Medium (with Hoechst 33258).
4. Wash 2–3 times with sterile PBS or normal saline for 3–5 minutes each time.
5. Examine directly under a fluorescence microscope without mounting.
Staining Results:
In apoptotic cells, the cell nuclei appear densely stained or show a densely stained fragmented pattern.
Precautions:
1. The concentration of Anti-fade Fluorescent Mounting Medium (with Hoechst 33258) is suitable for various routine staining applications.
2. Fluorescent dyes are susceptible to quenching; it is recommended to perform detection as soon as possible after staining.
3. Avoid repeated freeze-thaw cycles, as this may cause the reagent to lose efficacy.
4. Hoechst 33258 is mildly irritating to the human body; appropriate protective measures should be taken.
5. For your safety and health, wear a lab coat and disposable gloves during operation.
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