Hoechst 33342 is also known as Bisbenzimide H 33342 or HOE 33342. Its molecular formula is C₂₇H₂₈N₆O·3HCl·3H₂O, with a molecular weight of 615.99 and CAS number 23491‑52‑3. Hoechst 33342 is a cell‑permeable blue fluorescent dye with relatively low cytotoxicity. Hoechst 33342 staining is commonly used for apoptosis detection, and samples can be examined by fluorescence microscopy or flow cytometry after staining. It is also widely applied for routine nuclear staining or general‑purpose DNA staining.
The maximum excitation wavelength of free Hoechst 33342 is 346 nm and its maximum emission wavelength is 460 nm. After binding to double‑stranded DNA, its maximum excitation wavelength shifts to 350 nm and the maximum emission wavelength is 461 nm. This Hoechst 33342 staining solution can be directly used for nuclear staining of fixed cells or tissues, as well as for nuclear staining of live cells or tissues
Instructions for Use (For Reference):
1. For fixed cells or tissues
a. After fixation of cell or tissue samples, wash appropriately to remove fixatives. If immunofluorescence staining is required, perform immunofluorescence staining first, followed by Hoechst 33342 staining. If no additional staining is needed, proceed directly to Hoechst 33342 staining.
b. For adherent cells or tissue sections, add an appropriate volume of Hoechst 33342 staining solution to fully cover the sample. For suspension cells, add at least three times the sample volume of staining solution and mix well. Incubate at room temperature for 3‑5 min.
c. Aspirate the Hoechst 33342 staining solution and wash 2‑3 times (3‑5 min each time) with TBST, PBS or normal saline.
d. Observe samples directly under a fluorescence microscope or mount sections before observation. When apoptosis occurs, the nuclei of apoptotic cells show condensed fluorescence staining or fragmented condensed staining.
2. For live cells or cultured tissues
a. Add sufficient Hoechst 33342 staining solution to fully cover the samples to be stained. Generally, 1 mL staining solution is required per well of a 6‑well plate, and 100 μL per well of a 96‑well plate.
b. Incubate for 20‑30 min at the optimal cell‑culture temperature. Discard the staining solution and wash 2‑3 times with PBS or culture medium before fluorescence detection. Under fluorescence microscopy, apoptotic nuclei exhibit condensed or fragmented condensed staining.
Precautions:
1. Photobleaching occurs with all fluorescent dyes. It is recommended to complete detection on the same day after staining. Live‑cell or live‑tissue samples should be observed immediately after staining.
2. Anti‑fade mounting medium can be used to slow down photobleaching. Anti‑fade mounting medium (DW2004) can be ordered from Duowo.
3. Hoechst 33342 is harmful to humans. Handle with care and take adequate protective measures to avoid skin contact or inhalation.
4. For scientific‑research‑use‑only by qualified personnel. Not for clinical diagnosis or therapy, and not for food‑ or pharmaceutical‑related applications.
5. Wear a laboratory coat and disposable gloves throughout the procedure for your safety and health.
