Product Overview
DAPI is used for fluorescence imaging of cell nuclei.
Description
The blue fluorescent DAPI nucleic acid stain preferentially stains dsDNA, and the combination of DAPI and dsDNA will produce about 20-fold fluorescence enhancement. The blue fluorescence of DAPI is in sharp contrast to the green, yellow or red fluorescent probes of other structures. The counterstaining protocol is compatible with a wide range of cytological labeling techniques-antibody-based direct or indirect detection methods, mRNA in situ hybridization, or staining with fluorescent reagents specific to cellular structures.
Characteristic
Fluorescence excitation and emission profiles of DAPI bound to dsDNA.
Usage
Nuclear fluorescent probe
Stability
Stable under normal conditions.
Storage
2-8℃, protect from light.
Introduction
DAPI is a popular nuclear and chromosome counterstain, showing a distinct banding pattern in chromosomes. DAPI emits blue fluorescence upon binding to the minor groove of dsDNA with a 20-fold fluorescence enhancement. DAPI is provided as a 5 mg solid and a 5 mg/mL aqueous solution.
Handling
Always wear recommended Personal Protective Equipment. No special handing advice is required.
Category
Plant Organelle Fluorescent Probes
Excitation/Emission (nm)
358/461
Molecular Formula
C16H17Cl2N5
Counterstaining Cells in Suspension for Flow Cytometry
Sample Preparation
Use the fixation protocol appropriate for your sample, or use the following protocol.
1. Collect a cell suspension of 2 × 105 to 1 × 106 cells.
2. Pellet the cells by centrifugation and discard the supernatant.
3. Tap the tube to resuspend the pellet in the residual liquid and add 1 mL of PBS at room temperature.
4. Transfer the full volume of resuspended cells to 4 mL of absolute ethanol at -20℃ by pipetting the cell suspension slowly into the ethanol while vortexing at top speed. Leave the cells in ethanol at -20℃ for 5-15 minutes.
5. Pellet the cells by centrifugation and discard the ethanol.
6. Tap the tube to loosen the pellet and add 5 mL of PBS at room temperature. Allow the cells to rehydrate for 15 minutes.
Counterstaining Protocol
1. Prepare the DAPI stock solution by dissolving 5 mg DAPI in 1 mL dH2O to give 14.3 mM DAPI stock solution.
2. Dilute the DAPI stock solution to 3 μM in staining buffer
(100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% Nonidet P-40). A 1 mL volume will be required for each cell sample.
3. Centrifuge the cell suspension (from step 2.6) and discard the supernatant. Tap to loosen the pellet and add 1 mL of DAPI diluted in staining buffer.
4. Incubate for 15 minutes at room temperature.
Chromosome FISH Counterstaining
Sample Preparation
Prepare the specimen according to standard procedures. Briefly rinse the final preparations in dH2O before counterstaining to remove residual buffer salts from the slide. This final rinse will help reduce nonspecific background staining on the glass. Allow the preparation to air dry.
Counterstaining Protocol
1. Prepare the DAPI stock solution by dissolving 5 mg DAPI in 1 mL dH2O to give 14.3 mM DAPI stock solution.
2. Dilute the DAPI stock solution to 30 nM in PBS. Pipet 300 μL of this staining solution directly onto the specimen. A plastic coverslip can be used to distribute the dye evenly on the slide.
3. Incubate the specimen in the dark for 30 minutes at room temperature.
4. Carefully remove the coverslip and rinse the specimen briefly with PBS or dH2O to remove unbound dye.
5. Remove excess liquid from the slide by gently blotting around the sample with an absorbent tissue.
6. Place a glass coverslip on the slide and seal the edges with wax or nail polish. Alternatively, the preparation can be mounted in an antifade reagent according to the manufacturer’s directions.
7. View the sample using a fluorescence microscope with appropriate filters.
Full Name
DAPI (4',6'-Diamidino-2-phenylindole, dihydrochloride)
Potential Health Hazard Effects
May cause eye irritation in susceptible persons.
May cause skin irritation in susceptible persons.
May be harmful by inhalation.
May be harmful if swallowed.
Sensitization
R43 - May cause sensitization by skin contact.
Polymerization
Hazardous polymerization does not occur.
Notice
Use the reagent as soon as possible after unpacking!
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Datasheet 