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Hoechst 33342 (Bisbenzimide H 33342, HOE 33342) The molecular formula is C27H28N6O·3HCl, the molecular weight is 561.93, and the CAS number is 23491-52-3, which is a blue fluorescent dye that penetrates the cell membrane to the nucleus DNA. The staining fluid is weak in the solution, but the fluorescence reinforcement is bonded to the DNA small groove in the AT sequence enriched region, and thus is used for nuclear staining, or conventional DNA staining.
This product is a solution, and the Hoechst 33342 concentration is 1 mg / ml for staining, and the commonly used work concentration is recommended from 0.5 to 10 μg / ml. The maximum excitation wavelength of Hoechst 33342 is 346 nm, the maximum emission wavelength is 460 nm; after the binding with the double-stranded DNA, the maximum excitation wavelength is 350 nm, the maximum emission wavelength is 461 nm. The nucleus can be used for a fixed or non-fixed cell or tissue nucleus Mark, and can be tested by fluorescent microscope or Flow cytometer etc.
Storage and transportation
Ice bag (Wet ICE) transportation; -20 ° C protected from light, 12 months effective.
1. Dilute Hoechst 33342 staining solution (1 mg/mL) with PBS or other suitable buffer to 0.5-10 μg/mL Hoechst 33342 staining working solution;
2. For fixed cells or tissue sections:
a. For fixed cell or tissue samples, remove the fixative after fixation, and wash 2-3 times with PBS or other suitable buffer for 3-5 min each time (for detecting suspended cells, please follow the normal operation method for suspended cells , All experiments need to add centrifugation and other steps);
b. (Optional step) If you need to perform immunofluorescence staining on fixed cells or tissues, prioritize related treatments, and then perform Hoechst 33342 staining after treatment;
c. Take an appropriate amount of Hoechst 33342 staining working solution to stain the fixed cells or tissues, cover the sample and incubate at room temperature for about 5 minutes;
d. After removing Hoechst 33342 staining working solution, wash 2-3 times with PBS or other suitable buffer, each time for 3-5 min;
e. After mounting the slide or directly observe under a fluorescence microscope, the excitation wavelength is 350 nm, and the emission wavelength is 460 nm.
3. For living cells or cultured tissues:
a. Add an appropriate amount of Hoechst 33342 staining solution to the cell culture, about 1/10 of the volume of the cell culture medium, which must fully cover the sample to be stained; usually 1 mL staining solution is added to one well of the six-well plate, and one well of the 96-well plate Add 100 μL of staining solution to the medium; incubate in a 37°C incubator for 10-20 min;
b. After removing Hoechst 33342 staining working solution, wash 2-3 times with PBS or other suitable buffer, each time for 3-5 min;
c. Observe directly under a fluorescence microscope, the excitation wavelength is 350 nm, and the emission wavelength is 460 nm.
1. The concentration and time of incubation of Hoechst 33342 staining working solution can be adjusted according to the actual situation.
2. Fluorescent dyes have quenching problems. Try to complete the test on the same day after dyeing. Anti-fluorescence quenching mounting tablets (recommended G1401) can be used to slow down fluorescence quenching.
3. Please wear lab coat and disposable gloves during operation.