DAPI Nucleic Acid Stain

实验概要

The  blue-fluorescent DAPI nucleic acid stain preferentially stains dsDNA; it  appears to associate with AT clusters in the minor groove. Binding of  DAPI to dsDNA produces a ~20-fold fluorescence enhancement, apparently  due to the displacement of water molecules from both DAPI and the minor  groove. DAPI also binds RNA, however in a different binding mode—one  thought to involve AU-selective intercalation. The DAPI/RNA complex  exhibits a longer-wavelength fluorescence emission maximum than the  DAPI/dsDNA complex (~500 nm versus ~460 nm) and a quantum yield that is  only about 20% as high.
DAPI is a popular nuclear counterstain for  use in multicolor fluorescent techniques. Its blue fluorescence stands  out in vivid contrast to green, yellow, or red fluorescent probes of  other structures. When used according to our protocols, DAPI stains  nuclei specifically, with little or no cytoplasmic labeling. Both DAPI  and DAPI dilactate work well in these protocols. The DAPI dilactate form  may be somewhat more water soluble. The counterstaining protocols are  compatible with a wide range of cytological labeling techniques—direct  or indirect antibodybased detection methods, mRNA in situ hybridization,  or staining with fluorescent reagents specific for cellular structures.  DAPI can also serve to fluorescently label cells for analysis in  multicolor flow cytometry experiments. The following protocols can be  modified for tissue staining or for staining unfixed cells or tissues.

主要试剂

Materials Required but Not Provided
1. For fluorescence microscopy

1) PBS

2) Optional: antifade reagent

2. For flow cytometry

1) PBS

2) Absolute ethanol

3) Staining buffer (see step 3.1)

3. For chromosome FISH staining

1) PBS

2) dH₂O

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  (dH₂O) 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 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.
 
    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 dH₂O 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.