Ex/Em=350/461 nm (unbound DNA), Ex/Em=346/460 nm (bound DNA)

Hoechst 33342

C27H28N6O3HCl3H2O

Soluble in water

1. Preparation of Hoechst 33342 staining solution
(1) Add 1 mL of deionized water to the EP tube to prepare a 10 mg/mL Hoechst 33342 storage solution.
(2) Hoechst 33342 storage solution was diluted with PBS at a ratio of 1:2,000 to Hoechst 33342 working solution at a final concentration of 5 ug/mL.
2. Staining
(1) For fixed cells or tissues
a. For cell or tissue samples, after fixation, the fixative was removed by appropriate washing. If immunofluorescence staining was to be performed, Hoechst 33342 staining was performed after the completion of staining.
b. For adherent cells or tissue sections, a small amount of Hoechst 33342 staining solution was added to cover the sample. For suspended cells, at least three times the volume of the sample to be stained was added and mixed.
c. Remove Hoechst 33342 staining solution, wash the cells with TBST, PBS or normal saline for 2-3 times, 3-5 min each time.
Note: Washing is optional but not required, dyeing will not be affected after washing.
d. Observe directly under fluorescence microscope or observe under fluorescence microscope after mounting. When apoptosis occurs, the nuclei of apoptotic cells are dense and densely stained, or fragmented and densely stained.
(2) For living cells or tissues
a. Appropriate amount of Hoechst 33342 staining solution was added to cover the sample. Generally, 1 mL was added to 6-well plate per well, 100 uL to 96-well plate per well.
b. Incubate at room temperature 10-30 min, protected from light.
c. Remove Hoechst 33342 staining solution, wash the cells with PBS for 2-3 times, then add 50 uL PBS to cover the cells.
Observe directly under fluorescence microscope