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DAPI Nucleic Acid Stain

互联网2013-11-13

1369

实验试剂

Material	Amount	Storage	Stability
DAPI dihydrochloride (MW = 350.3)	10 mg	Room temperature Protect from light	When stored as directed, product solids should be stable for at least 1 year.
DAPI dilactate (MW = 457.5)	10 mg	Room temperature Protect from light	When stored as directed, product solids should be stable for at least 1 year.
DAPI dihydrochloride, FluoroPure™ grade (≥98% pure)	10 mg	Room temperature Protect from light	When stored as directed, product solids should be stable for at least 1 year.
	10 mg	Room temperature Protect from light	When stored as directed, product solids should be stable for at least 1 year.

Materials Required but Not Provided
1. For fluorescence microscopy

2) Optional: antifade reagent

2. For flow cytometry

2) Absolute ethanol

3) Staining buffer (see step 3.1)

3. For chromosome FISH staining

3) Wax or nail polish

4) Optional: antifade reagent

Preparing the DAPI Stock Solution
To make a 5 mg/mL DAPI stock solution (14.3 mM for the dihydrochloride or 10.9 mM for the dilactate), dissolve the contents of one vial (10 mg) in 2 mL of deionized water (dH₂ O) or dimethylformamide (DMF). The less water-soluble DAPI dihydro-chloride may take some time to completely dissolve in water and sonication may be necessary.
Note: Neither of these DAPI derivatives is very soluble in phosphate-buffered saline (PBS). For long-term storage the stock solution can be aliquoted and stored at ≤–20°C. For shortterm storage the solution can be kept at 2–6°C, protected from light. When handled properly, DAPI solutions are stable for at least six months.

实验步骤

1. Counterstaining Adherent Cells for Fluorescence Microscopy
1) Sample Preparation

Use the fixation protocol appropriate for your sample. DAPI staining is normally performed after all other staining. Note that fixation and permeabilization of the sample are not necessary for counterstaining with DAPI.
2) Counterstaining Protocol

A. Equilibrate the sample briefly with phosphate-buffered saline (PBS).

B. Dilute the DAPI stock solution to 300 nM in PBS. Add approximately 300 μL of this dilute DAPI staining solution to the coverslip preparation, making certain that the cells are completely covered.

C. Incubate for 1–5 minutes.

D. Rinse the sample several times in PBS. Drain excess buffer from the coverslip and mount. We recommend using a mounting medium with an antifade reagent such as our SlowFade ® Gold antifade reagent (S36936) or ProLong ® Gold antifade reagent (P36930).

E. View the sample using a fluorescence microscope with appropriate filters.

2. Counterstaining Cells in Suspension for Flow Cytometry
1) Sample Preparation

Use the fixation protocol appropriate for your sample, or use the following protocol.

A. Collect a cell suspension of 2 × 10⁵ to 1 × 10⁶ cells.

B. Pellet the cells by centrifugation and discard the supernatant.

C. Tap the tube to resuspend the pellet in the residual liquid and add 1 mL of PBS at room temperature.

D. Transfer the full volume of resuspended cells to 4 mL of absolute ethanol at –20°C by pipetting the cell suspension slowly into the ethanol while vortexing at top speed. Leave the cells in ethanol at –20°C for 5–15 minutes.

E. Pellet the cells by centrifugation and discard the ethanol.

F. Tap the tube to loosen the pellet and add 5 mL of PBS at room temperature. Allow the cells to rehydrate for 15 minutes.

2) Counterstaining Protocol

A. 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.

B. 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.

C. Incubate for 15 minutes at room temperature.

D. Analyze by flow cytometry in the presence of the dye. If the cells are to be viewed by fluorescence microscopy, centrifuge the sample, remove the supernatant and resuspend cells in fresh buffer. Apply a drop of the suspension to a microscope slide, cover with a coverslip and view.

3. Chromosome FISH Counterstaining
1) Sample Preparation

Prepare the specimen according to standard procedures.5,6 Briefly rinse the final preparations in dH₂ O 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.

2) Counterstaining Protocol

A. 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.

B. Incubate the specimen in the dark for 30 minutes at room temperature.

C. Carefully remove the coverslip and rinse the specimen briefly with PBS or dH₂ O to remove unbound dye.

D. Remove excess liquid from the slide by gently blotting around the sample with an absorbent tissue.

E. Place a glass coverslip on the slide and seal the edges with wax or nail polish. Alternatively, the preparatio can be mounted in an antifade reagent according to the manufacturer’s directions.

F. View the sample using a fluorescence microscope with appropriate filters.

注意事项

DAPI is a known mutagen and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations.

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